RESUMO
The objective of this study was to investigate the changes in the functional properties (pH, water holding capacity [WHC], water binding capacity [WBC], cooking losses [CL], defrosting losses [DL]), color parameters (L*, a*, b*, C, h°, ΔE), and sensory evaluation of breast (BM) and leg (LM) muscles from 17-wk-old female White Koluda geese packaged in a vacuum and stored in frozen conditions at -20°C. During 17 wk, the geese were fed ad libitum on the same complete feed. The samples (18 BM and 18 LM) from the right part of the carcasses were stored for 30, 90, 80, 270, and 365 d. The changes in functional properties were established using a standard method used in the meat industry, according to Wierbicki et al. (1962), Grau and Hamm (1953), and CIE, (1986). Sensory evaluation was established according to defined parameters in PN-ISO 8586-2:2008. The time of frozen storage affected the decrease in WHC and WBC of BM and LM. Moreover, the LM can be characterized by a higher WHC and WBC compared to the values in the BM. It was established that CL and DL, which are the critical quality indicators, negatively increased in BM and LM during frozen storage. Considering the sensory evaluation and L*, a*, b*, C, it was established that changes in BM and LM during frozen storage were unfavorable. The scores given for smell, taste, consistency, and general appearance, as also L*, a*, and b* parameters decreased significantly during frozen storage. In addition, BM received lower scores for general appearance (at 180th and 270th day), and L* (in all frozen storage), than LM. BM and LM characterized the parameter ΔE in the range of 0.44 to 1.45, which allowed us to conclude that slight color differences were visible in these muscles (<2). Based on the study, it can be suggested that the optimal frozen storage time for BM and LM should not be longer than 180 d.
Assuntos
Galinhas , Gansos , Feminino , Animais , Gansos/fisiologia , Músculo Esquelético/metabolismo , Carne/análise , Água/metabolismoRESUMO
The effect of sous-vide (S-V), microwave cooking (M) and stewing (S) on the fatty acid profile and lipid indices of goose meat was investigated. The research material involved breast muscles (24 with skin and 24 without skin) cut from carcasses of 17-week-old females "Polish oat geese." Gas chromatography was used to establish the fatty acid profile and lipid indices were calculated. The kind of heat treatment and the type of goose meat (muscles with- and without skin) affected the fatty acid profile and lipid indices. The sum of SFA was higher in S-V, M, and S samples for both kinds of meat than in raw ones. The cooked samples with skin had a lower percentage of Æ© SFA than the skinless meat. S-V and M cooking (for meat with skin) caused an increase, while in the case of S heating (for both kinds of meat) there were no significant differences in Æ© MUFA compared to raw samples. The S-V, M, and S meat with skin was characterized by a higher value of Æ© MUFA than skinless ones. The Æ© PUFA was lower in S-V and M than in raw meat, wherein this decline was higher for M ones (for both kinds of meat). The M meat with skin had the lowest and S without skin the highest share of Σ PUFA. Heat treatment caused an increase in Σ PUFA n-6/n-3 ratio, the lowest value was shown by the S-V muscles. Sous-vide cooking was more beneficial for consumers than the remaining methods in terms of Σ DFA/Σ OFA, Σ UFA/Σ SFA, NVI, health-promoting index (HPI), inflammatory biomarker indexes, and Σ SFA, Σ OFA, Σ UFA, Σ DFA values for meat with skin (but not all of these were significant). In turn, stewing of meat without skin was more favorable than S-V and microwave cooking in relation to indexes such as: Σ DFA/Σ OFA, Σ UFA/Σ SFA, Σ PUFA/Σ SFA, PI, UI, AI, TI, HPI.
Assuntos
Ácidos Graxos , Gansos , Feminino , Animais , Ácidos Graxos/análise , Micro-Ondas , Galinhas , Carne/análise , Culinária/métodosRESUMO
The objective of this study was to investigate the changes of the fatty acid in breast (BM) and leg (LM) muscles from 17-wk-old female White Koluda geese packaged in a vacuum and stored in freezing conditions at -20°C. During 17 weeks, the geese were fed ad libitum on the same complete feed. The samples (18 LM and 18 BM) from the right part of the carcasses were stored for 30, 90, 80, 270, and 365 d. The changes in the fatty acid profile were established by gas chromatography. In this work, there were also calculated lipid profile indicators such as Σ PUFA n-6/Σ PUFA n-3, Σ UFA/Σ SFA, and Σ PUFA/Σ SFA. Time of frozen storage affected the decrease in Σ SFA, Σ MUFA, and Σ PUFA of BM and LM. The statistical analysis of the obtained data shows that the type of muscle also generally affected the fatty acid profile. The BM are characterized higher proportion of Σ SFA, and the LM are defined as containing more Σ MUFA and Σ PUFA. Extending frozen storage time caused only the deterioration of Σ PUFA n-6/Σ PUFA n-3. The Σ PUFA n-6/Σ PUFA n-3 were the highest in BM and LM on the 365th day of storage. Although the Σ PUFA n-6/Σ PUFA n-3 ratio in muscles stored for 180, 270, and 365 d was higher than the recommended values. The lipid profile indicators (Σ UFA/Σ SFA, and Σ PUFA/Σ SFA) were similar in raw meat and in all frozen storage samples. It means that frozen storage didn't affect this index and the BM and LM have the same quality from the dietary point of view. Leg muscles during frozen storage are characterized by higher Σ UFA/Σ SFA and Σ PUFA/Σ SFA than the breast muscles.
Assuntos
Ácidos Graxos , Gansos , Feminino , Animais , Ácidos Graxos/análise , Gansos/fisiologia , Congelamento , Galinhas , Carne/análiseRESUMO
The objective of this study was to investigate the oxidation activity and lipid oxidation changes in breast (BM) and leg (LM) muscles from 17-wk-old female White Koluda geese packaged in a vacuum, and stored in freezing conditions at -20°C. The geese were fed ad libitum during the experimental period (up to 17 wk) on the same complete feed. The samples of LM (n = 18) and BM (n = 18) from the right carcass were stored for 30, 90, 80, 270, and 365 d. Lipid oxidation was described by determining changes in: TBARS value expressing the amount of malondialdehyde (MDA), total antioxidant capacity (TAC) measured by DPPH and ABTS methods and total reduction potential (TRP) measured by FRAP method. Moreover, total heam pigments pigment (THP), relative concentration of myoglobin (Mb), oxymyoglobin (MbO2), and metmyoglobin (MMb) were determined in this study. Time of storage affected the TAC, TRP, TBARS, and the color stability of BM and LM. The THP concentration and Mb proportion decrease gradually during the 365 d of frozen storage, while the relative concentration of MMb increase in BM and LM. It was noted that the shares of MMb in both analyzed types of muscles stored for 365 d did not exceed 0.4. This value is considered to be the limit, after which the meat takes on an intense gray-brown color not accepted by the consumer. The oxidation processes occurring during frozen storage caused an increase of TBARS and a decrease of DPPHâ¢, ABTSâ¢+, FRAP values in both kinds of muscles. The amounts of TBARS during frozen storage of muscles did not exceed 2.0 mg MDA/kg of meat. A higher value of TBARS than 2.0 causes a lack of acceptance by consumers of the flavor profile. Based on the results concerning changes in the heam pigments, as well as changes related to the oxidation of lipids, we cannot unequivocally state in which types of muscles the changes had a more intense course.
Assuntos
Galinhas , Gansos , Animais , Feminino , Congelamento , CarneRESUMO
The effect of water bath cooking (WBC), oven convection roasting (OCR), grilling (G), pan-frying (PF) on the fatty acid profile and health lipid indices of goose meat was investigated in this study. The experimental material covered 80 breast muscles (40 with skin and subcutaneous fat and 40 without skin) cut from carcasses of 17-week-old "Polish oat geese". The fatty acid profile of meat was determined by gas chromatography and health lipid indices were calculated. It was stated that the kind of heat treatment as well as the type of goose meat (muscles with and without skin) affected the fatty acid profile and health lipid indices. The sum of SFA was significantly higher in cooked samples for both kinds of meat than in raw ones. The cooked samples with skin had a lower increase in Æ© SFA than the skinless meat. Boiling (meat without skin) and pan-frying (both kinds of meat) caused a slight decrease, while grilling and oven convection roasting (both kinds of meat) caused an increase of Æ© MUFA in comparison to raw samples. Moreover, meat with skin is characterized by a higher value of Æ© MUFA than meat without skin for all cooking methods. The Æ© PUFA was lower in all cooked samples than in raw meat, wherein this decline was usually higher for skinned meat. The G meat was the lowest and PF the highest in Σ PUFA for both kinds of meat after heat treatment. The highest loss showed C20:4 n-6 in OCR samples and the lowest C18:2 n-6 in PF (both kinds of meat). Heat treatment caused an increase in the Σ PUFA n-6/n-3 ratio, wherein the lowest value was shown by the WBC samples without skin, and the highest by OCR with skin. Water bath cooking of meat was more beneficial for consumers in terms of AI, TI, Σ DFA/Σ OFA, Σ PUFA/Σ SFA, Σ UFA/Σ SFA indexes and Σ SFA, Σ OFA values than the remaining methods.
Assuntos
Ácidos Graxos , Gansos , Animais , Galinhas , Temperatura Alta , Lipídeos , Carne/análise , PolôniaRESUMO
The effect of water bath cooking (WBC), oven convection roasting (OCR), grilling (G), pan frying (PF) on selected physical properties of goose meat was compared in this study. A measurement of cooking loss, texture, color parameters, and sensory evaluation was carried out. The experimental material covered 96 breast muscles cut from carcasses of 17-week-old "Polish oat geese." The kind of goose meat (with and without skin) and the type of heat treatment affected cooking loss, shear force (SF), and rheological parameters (hardness, cohesiveness, gumminess, and chewiness). The water bath-cooked and pan-fried samples for both kinds of meat were characterized by lower cooking loss than other ones. Goose meat with skin and subcutaneous fat showed higher cooking loss and lower SF value, hardness, gumminess, and chewiness than that without skin for all methods. The water bath-cooked samples were characterized by the lowest SF value, hardness, and chewiness for both kinds of meat. They had the highest value of L∗ parameter and were characterized by a lighter color among others, too. Pan-fried meat showed the highest value of a∗ and lowest of ho parameters; the color of these samples was redder. Moreover, the lower C values of oven convection-roasted and grilled samples showed that they were brighter. According to the Comission Internationale de l'Eclairage classification, the ΔE parameter only for G and OCR indicated noticeable color differences (<2), whereas other pairs had visible differences. The method of cooking affected sensory descriptors such as the intensity of flavor and aroma, tenderness, juiciness, springiness, cohesiveness, and overall palatability of goose meat. The goose samples of PF, G, and OCR were characterized as very good and WBC as extremely desirable overall palatability. However, in the next stage of research, there is a need to study changes in the chemical composition, the degree of lipid oxidation, and the nutritional value of this meat that underwent different methods of cooking. Only then it will be possibly to clearly determine which method of the heat treatment of goose meat is optimal.
Assuntos
Gansos , Temperatura Alta , Carne , Animais , Culinária , Carne/análise , Carne/normasRESUMO
The objective of the work was to investigate the color and lipid oxidation changes of goose breast meat packaged in vacuum and modified atmosphere (MA) conditions consisting of 80% O2, 20% CO2, and stored in refrigerated conditions at 4°C. Color stability was monitored by determining total heme pigments concentration; relative concentration of myoglobin, oxymyoglobin, and metmyoglobin; parameters of color L*, a*, b*, and sensory evaluation of the surface color. Lipid stability was measured by determining thiobarbituric acid reactive substances (TBARS). The samples were examined in 24 h after slaughter (unpacked muscles) and on d 4, 7, 9, 11 of storage (muscles packed in vacuum and in MA). Through the time of storage, samples packed in MA had higher TBARS values in comparison to the meat packed in vacuum. For samples packed in two types of atmospheres, the total pigments concentration decreased gradually within 11 d of storage. It was observed that relative metmyoglobin concentration increased whereas relative oxymyoglobin concentration decreased in total heme pigments in the MA stored muscle. The relative concentration of all three myoglobin forms sample packed in vacuum remained unchanged. The color parameters (L*, a*, b*) did not change for 11 d of storage for the vacuum packed meat. The value of the color parameter a* decreased and the value of the color parameters L* and b* increased in the samples packaged in MA. The data prove that if you store goose meat in MA (consisting of 80% O2, 20% CO2) or vacuum, the unchanged surface color is preserved for 9 and 11 day, respectively.Vacuum appears to be a better method as regards the maintaining of lipid stability in goose meat.
Assuntos
Embalagem de Alimentos/normas , Metabolismo dos Lipídeos , Carne/análise , Vácuo , Animais , Pressão Atmosférica , Cor , Conservação de Alimentos , Gansos/fisiologia , Músculos Peitorais/fisiologiaRESUMO
The objective of the work was to characterize the color of the of the goose breast meat packaged in protective atmosphere and stored in the refrigerated conditions. The aim was realized by determination of total heme pigment concentration; relative concentration of myoglobin, oxymyoglobin, and metmyoglobin; parameters of color L* (lightness), a* (redness), and b* (yellowness); and sensory evaluation of the surface color. The experimental material was White Koluda goose boneless breast meat with the skin from industrial slaughter. The following 2 protective atmospheres were used in the study: vacuum and modified atmosphere (MA) consisting of 80% O2 and 20% CO2. The muscles packed in protective atmosphere were examined on d 4, 7, 11, and 14 of storage. A control sample was goose breast meat stored in air and tested after 24 h after slaughter. The total pigment concentration decreased gradually within 14 d of storage for samples packed in 2 types of atmospheres. The increase in relative concentration of metmyoglobin and the decrease in oxymyoglobin relative concentration in total heme pigments in the meat stored in MA was noticed. However, in all times of storage, the relative concentration of the 3 samples of myoglobin forms stored in vacuum was unchanged. The color parameters (L*, a*, b*) did not change for 14 d of storage in the muscles packed in vacuum. One can state a decrease of the value of the color parameter a* as well as an increase of the value of the color parameter b* in the samples packed in MA. From d 11 to 14 of storage, goose meat packed under MA had lower sensory evaluation intensity of color than muscles under vacuum. The obtained data indicated that the surface color of goose breast meat packed in MA (consisting of 80% O2, 20% CO2) or vacuum packed was maintained for 11 and 14 d, respectively.
Assuntos
Dióxido de Carbono/química , Embalagem de Alimentos , Armazenamento de Alimentos/métodos , Carne/normas , Oxigênio/química , Animais , Anseriformes , Atmosfera , OxirreduçãoRESUMO
The aim of the study was to compare the chemical and amino acid composition of breast (pectoralis major) and thigh (biceps femoris) muscles in 17-wk-old geese from 2 Polish conservative flocks: Rypinska (Ry, n = 20) and Garbonosa (Ga, n = 20). The geese were fed ad libitum during the experimental period on the same complete feed. Genotypes affected the moisture and fat content of breast and thigh meat. The Ga geese were characterized by higher moisture as well as lower fat lipid content compared with the Ry breast and thigh muscles. The amino acid proportions of meat proteins depended on the goose flock and type of muscles, where significant differences were found. The proteins of Ga breast muscles contained more glutamic acid, glycine, lysine, tryptophan, histidine, and methionine, and less aspartic acid, proline, serine, leucine, valine, phenyloalanine, tyrosine, and threonine than the Ry geese (P ≤ 0.05). The proteins of Ry thigh muscles were characterized by higher content of proline, serine, and essential amino acids (without lysine and methionine) and lower glutamic and asparagine acid, alanine, and glycine compared with the Ga flock. According to the Food and Agriculture Organization of the United Nations/World Health Organization (1991) standard, tryptophan was the amino acid limiting the nutritional value of meat proteins of Ry breast muscles (amino acid score for tryptophan = 90%). Except for tryptophan, the meat proteins of the investigated raw materials contained more essential amino acids than the standard. The total content of essential amino acids for all investigated muscles was also higher (52.51 to 55.54%) than the standard (33.90%). It is evident that muscle protein from both flocks of geese have been characterized by high nutritional value. The values of the essential amino acid index of breast muscle proteins were similar in both flocks.
Assuntos
Aminoácidos/análise , Gansos/fisiologia , Carne/análise , Aminoácidos Essenciais/análise , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Gansos/genética , Carne/normas , Músculos Peitorais/química , Polônia , Músculo Quadríceps/químicaRESUMO
Male Wistar rats were divided into two groups. Rats of group 1 were fed basal powdered diet containing 610 ppm 2,4- diaminoanisole sulfate (DAAS), 46 ppm 4,4'-thiodianiline (TDA) and 200 ppm N,N'-diethylthiourea (DETU) for 52 weeks (DTD treatment). Rats of group 2 were maintained on basal diet throughout the experiment as controls. At 52 weeks all surviving rats were sacrificed and subjected to an autopsy. Thyroid, lungs, stomach, liver, spleen, kidneys, testes and all gross lesions suspected of being a tumor were removed. After DTD treatment, the incidence of thyroid hyperplasia and papillary thyroid carcinoma was 59% (10/17) and 65% (11/17), respectively. Hepatocellular adenoma was induced in 2 of 17 rats (12%). Papillary thyroid carcinoma metastasis was found in the lung of 1 rat. No neoplastic tumors were found in kidney, spleen, stomach and testis tissue.
Assuntos
Compostos de Anilina/toxicidade , Carcinógenos/toxicidade , Neoplasias Experimentais/induzido quimicamente , Fenilenodiaminas/toxicidade , Tioureia/análogos & derivados , Tioureia/toxicidade , Adenoma de Células Hepáticas/induzido quimicamente , Adenoma de Células Hepáticas/patologia , Animais , Carcinoma Papilar/induzido quimicamente , Carcinoma Papilar/patologia , Carcinoma Papilar/secundário , Sinergismo Farmacológico , Hiperplasia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Neoplasias Experimentais/patologia , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Especificidade da Espécie , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/induzido quimicamente , Neoplasias da Glândula Tireoide/patologiaRESUMO
The application of the oxathiaphospholane approach for the synthesis of dithymidine boranphospate was evaluated. It was shown, that although the nucleoside-3'-O-oxathiaphospholane-borane complexes 2 or 6 could not be chromatographically separated into diastereomerically pure species due to their apparent instability to moisture, they can be successfully applied to the non-stereocontrolled formation of internucleotide boranophosphate bond by reaction with 5'-OH-nucleoside in the presence of DBU. Attempts to apply the related dithiaphospholane approach for the preparation of dithymidine boranophosphorothioate were unsuccessful.
Assuntos
Compostos de Boro/síntese química , Nucleotídeos/química , Ribose/análogos & derivados , Ribose/química , Timidina/análogos & derivados , Timidina/síntese química , Modelos Químicos , Nucleotídeos/síntese química , Fosfatos/químicaRESUMO
The application of the oxathiaphospholane approach for the stereocontrolled synthesis of LNA dinucleoside phosphorothioate is described. The reaction of ring opening condensation proceeds in CH3CN solution in high yield and with over 96% stereoselectivity. One of diastereomers of LNA dinucleoside phosphorothioate (presumably R(P)) was found to be readily digested by svPDE.
Assuntos
Nucleotídeos/síntese química , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Fosfatos/síntese química , Diester Fosfórico Hidrolases/metabolismo , Ribose/síntese química , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Fosfodiesterase I , Ribose/análogos & derivados , Ribose/metabolismo , EstereoisomerismoRESUMO
The cytotoxicity of anti-PAI-5 hexadecanucleotides (phosphodiesters and phosphorothioates) and their conjugates with lipophilic alcohols was tested in EA.hy 926 hybrid endothelial cells. Some cytotoxicity was found for cholesteryl and bornyl conjugates at concentrations higher than those used for antisense inhibition experiments.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Oligonucleotídeos/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Linhagem Celular , Humanos , Oligonucleotídeos/químicaRESUMO
A series of conjugates containing residues of lipophilic alcohols covalently bound to 5' end of oligodeoxyribonucleotides targeted against human plasminogen activator inhibitor (PAI-1) mRNA was synthesized via the oxathiaphospholane approach. The highest anti-PAI-1 activity in EA.hy 926 endothelial cell cultures was found for conjugates containing menthyl or heptadecanyl groups linked with an oligonucleotide complementary to a segment of human PAI-1 mRNA. The phosphodiester antisense oligonucleotides, which otherwise exhibit only limited anti-PAI-1 activity, were found to be more active than phosphorothioate oligonucleotides when conjugated to lipophilic alcohol residues. For menthyl conjugates an evidence of antisense mechanism of inhibition was found.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , Humanos , Oligodesoxirribonucleotídeos Antissenso/síntese química , Oligodesoxirribonucleotídeos Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Thymidine-5'-fluorothiophosphate, dTMP(S)-F, was synthesized by the oxathiaphospholane, and thymidine 5'-dithiophosphate, dTMPS2, by the dithiaphospholane, method. To estimate the role of 5'-phosphate group ionization in binding of pyrimidine nucleotides by thymidylate synthase, dTMP(S)-F was studied as an inhibitor of mouse tumour (L1210) enzyme, and its inhibitory properties were compared with those of dTMPS2, a close dTMP analogue. While dTMPS2 proved to be an inhibitor, competitive vs dUMP, with K(i)app = 94 microM, the 5'-fluorothiophosphate congener displayed no activity, indicating that the enzyme requires for binding the presence of a dianionic 5'-phosphate group in a nucleotide.
Assuntos
Inibidores Enzimáticos/síntese química , Leucemia L1210/tratamento farmacológico , Organotiofosfatos/síntese química , Timidilato Sintase/antagonistas & inibidores , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/uso terapêutico , Camundongos , Organotiofosfatos/farmacologiaRESUMO
Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.
Assuntos
Oligonucleotídeos Antissenso/síntese química , Aminas/química , Sequência de Bases , Cromatografia Líquida de Alta Pressão/métodos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/isolamento & purificação , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , Compostos Organofosforados/isolamento & purificação , OxirreduçãoRESUMO
The effect of systemic inhibition of PAI-1 expression in rats by PS-16R, a phosphorothioate analogue of hexadecadeoxyribonucleotide complementary to a signal peptide coding sequence of rat PAI-1 mRNA, on PAI-1 activity in blood plasma and thrombus formation was studied in rat models for experimental thrombosis. In previous in vitro studies, oligonucleotides of PS-16R family have been shown to inhibit efficiently PAI-1 synthesis in endothelial cells by antisense mechanism. When PS-16R was administered intravenously as a single bolus injection (1 to 5 mg per rat), it produced a significant reduction in PAI-1 activity of blood plasma. This effect was both time- and concentration-dependent. Under the same conditions, three groups of rats were treated with control oligodeoxynucleotides such as PS-16R with double mismatches, with scrambled sequence, and an oligodeoxynucleotide with sense sequence (complementary to PS-16R), respectively. Based on these preliminary experiments, a low dose of 1.5 mg per rat was selected to produce approximately 20-30% reduction of PAI-1 activity in blood plasma and the effect of such a decrease in PAI-1 expression was tested on thrombus formation in two rat models for experimentally induced thrombosis. Such a limited decrease in PAI-1 activity produced a significant antithrombotic effect in the arterial thrombosis model. There was a profound delay in the occlusion time in rats treated with PS-16R when compared to control animals (80 +/- 3 and 55 +/- 3 h, respectively), although blood plasma activity of PAI-1 in the same groups of rats differed only by 20%. There was also a tendency to reduce both an incidence of venous thrombosis (58.33 and 68.11%, respectively) and thrombus weight (2.1 +/- 0.4 and 2.9 +/- 0.9 mg, respectively) in the animals treated with PS-16R. However, this effect was not significant. Thus, low dose of PS-16R through inhibition of PAI-1 synthesis in targeted cells in rats reduced PAI-1 activity in blood plasma and protected against arterial thrombus formation in the rat.
Assuntos
Fibrinólise/fisiologia , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Trombose/sangue , Trombose/fisiopatologia , Animais , Fibrinólise/efeitos dos fármacos , Oligonucleotídeos Antissenso/administração & dosagem , RNA Mensageiro/antagonistas & inibidores , RatosRESUMO
Nitric oxide (NO) plays an important role in cellular signaling and host defense, and it also contributes to the deleterious effects of immune response. Until recently, the lack of specific inhibitors of various forms of nitric oxide synthase (NOS) hampered a stringent evaluation of the role played by inducible NOS (iNOS) in cell damage. The present study investigated the use of antisense oligodeoxynucleotides (AS-ODNs) to selectively inhibit the expression of iNOS. AS-ODNs (1-10 microM) inhibited, in a time-dependent and dose-dependent manner, iNOS activity in RAW 264.7 murine macrophages. Maximal inhibitory effect was >90%, and control ODNs had little or no effect on NO production. Treatment with AS-ODNs decreased iNOS protein and mRNA level in studied cell, and control ODNs again were ineffective. The decreased levels of the target mRNA in AS-ODN-treated samples suggest that the AS-ODNs used act as substrates for ribonuclease (RNase) H. Lipofection enhanced the effect of AS-ODNs on iNOS activity. However, this potentiation appears to be different from the antisense effect, in which the AS-ODNs studied were involved. Liposaccharide/interferon-gamma (LPS/IFN-gamma) induced iNOS, and increased NO production impaired the viability of macrophages. Treatment of RAW 264.7 cells with 10 microM AS-ODNs prevented the NO-induced lethal cell damage.
Assuntos
Inibidores Enzimáticos/farmacologia , Macrófagos/efeitos dos fármacos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Animais , Linhagem Celular , Sinergismo Farmacológico , Indução Enzimática , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Fosfatidiletanolaminas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Hexadecadeoxyribonucleotides complementary to a fragment of human PAI-1 mRNA located upstream of the start codon and their phosphorothioate analogs were studied in cultured HUVECs as sequence-dependent inhibitors of PAI-1 expression. The activity of the random mixture of diastereomers of phosphorothioate hexadecanucleotide PS-16H has been compared with that of isosequential, stereoregular [All-Sp] and [All-Rp] isomers. The highest inhibitory effect on PAI-1 synthesis was observed with the [All-Sp] diastereomer. Stereorandom phosphorothioate oligonucleotide PS-16R complementary to the same region of rat PAI-1 mRNA, when injected into tail vein of rats, substantially decreased the level of PAI-1 in blood plasma.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio , Tionucleotídeos/farmacologia , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Humanos , Masculino , Compostos Organofosforados/química , Inibidor 1 de Ativador de Plasminogênio/genética , RNA Mensageiro/genética , Ratos , Tionucleotídeos/químicaRESUMO
Oligonucleotides with a nucleotide sequence complementary to various regions of human plasminogen activator inhibitor type-1 (PAI-1) mRNA have been studied as antisense inhibitors of expression of PAI-1 protein in cultured cells [human umbilical vein endothelial cells (HUVEC), human aortic smooth muscle cells, human hybrid endothelial cells]. Hexadeca(deoxyribonucleoside phosphorothioate) 13 complementary to a fragment of a signal peptide PAI-1 mRNA was found to be most active, giving ca. 70% inhibition of PAI-1 release in a time- and dose-dependent way. The stereo-regular All-S(P) and All-R(P) diastereomers of 13 were studied and found to inhibit PAI-1 synthesis in HUVEC in a stereo-dependent manner, with the All-S(P) diastereomer considerably more active than the stereo-random construct and All-R(P) isomer. The observed stereo-dependent activity of oligonucleotide phosphorothioate constructs is presumably governed by their resistance to nucleases. The corresponding phosphodiester analogue of 13 was not active unless covalently bound at its 5'-end to a lipophilic alcohol residue (menthol, heptadecanol). The observed antisense activity of phosphodiester oligonucleotide bioconjugates in cultured human hybrid endothelial cells was paralleled by their increased stability in human plasma with respect to unconjugated oligonucleotide. The oligo(deoxyribonucleoside phosphorothioate) complementary to the same signal peptide region of rat PAI-1 mRNA was found to reduce the PAI-1 level in blood plasma of rats after intravenous administration into the tail vein. The effect was both time- and dose-dependent. The same oligonucleotide was found to protect against arterial thrombus formation in the rat (lower incidence of venous thrombosis, lower thrombus weight, and increased occlusion time in experimentally induced thrombosis). An anti-PAI-1 inhibitory activity has been independently reported for a 20-mer oligo(2'-O-methyl-ribonucleoside phosphorothioate) complementary to a 3'-untranslated region of human PAI-1 mRNA in cultured HUVEC and human aortic smooth muscle cells.