Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

Modulation of ROS production in human leukocytes by ganglioside micelles

Recent studies have reported that exogenous gangliosides, the sialic acid-containing glycosphingolipids, are able to modulate many cellular functions. We examined the effect of micelles of mono- and trisialoganglioside GM1 and GT1b on the production of reactive oxygen species by stimulated human polymorphonuclear neutrophils using different spectroscopic methods. The results indicated that exogenous gangliosides did not influence extracellular superoxide anion (O2.-) generation by polymorphonuclear neutrophils activated by receptor-dependent formyl-methionyl-leucyl-phenylalanine. However, when neutrophils were stimulated by receptor-bypassing phorbol 12-myristate 13-acetate (PMA), gangliosides above their critical micellar concentrations prolonged the lag time preceding the production in a concentration-dependent way, without affecting total extracellular O2.- generation detected by superoxide dismutase-inhibitable cytochrome c reduction. The effect of ganglioside GT1b (100 µM) on the increase in lag time was shown to be significant by means of both superoxide dismutase-inhibitable cytochrome c reduction assay and electron paramagnetic resonance spectroscopy (P < 0.0001 and P < 0.005, respectively). The observed phenomena can be attributed to the ability of ganglioside micelles attached to the cell surface to slow down PMA uptake, thus increasing the diffusion barrier and consequently delaying membrane events responsible for PMA-stimulated O2.- production.

Effect of hyperoxia on the intestinal IgA secretory component in neonatal rats and on intestinal epithelial cells in vitro

Oxygen therapy is essential for the treatment of some neonatal critical care conditions but its extrapulmonary effects have not been adequately investigated. We therefore studied the effects of various oxygen concentrations on intestinal epithelial cell function. In order to assess the effects of hyperoxia on the intestinal immunological barrier, we studied two physiological changes in neonatal rats exposed to hyperoxia: the change in intestinal IgA secretory component (SC, an important component of SIgA) and changes in intestinal epithelial cells. Immunohistochemistry and Western blot were used to detect changes in the intestinal tissue SC of neonatal rats. To detect intestinal epithelial cell growth, cells were counted, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Giemsa staining were used to assess cell survival. Immunohistochemistry was used to determine SC expression. The expression of intestinal SC in neonatal rats under hyperoxic conditions was notably increased compared with rats inhaling room air (P < 0.01). In vitro, 40% O2 was beneficial for cell growth. However, 60% O2 and 90% O2 induced rapid cell death. Also, 40% O2 induced expression of SC by intestinal epithelial cells, whereas 60% O2did not; however, 90% O2 limited the ability of intestinal epithelial cells to express SC. In vivo and in vitro, moderate hyperoxia brought about increases in intestinal SC. This would be expected to bring about an increase in intestinal SIgA. High levels of SC and SIgA would serve to benefit hyperoxia-exposed individuals by helping to maintain optimal conditions in the intestinal tract.

Relationship between work rate and oxygen uptake in mitochondrial myopathy during ramp-incremental exercise

We determined the response characteristics and functional correlates of the dynamic relationship between the rate (&#916;) of oxygen consumption ( <img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1188">O2) and the applied power output (work rate = WR) during ramp-incremental exercise in patients with mitochondrial myopathy (MM). Fourteen patients (7 males, age 35.4 ± 10.8 years) with biopsy-proven MM and 10 sedentary controls (6 males, age 29.0 ± 7.8 years) took a ramp-incremental cycle ergometer test for the determination of the <img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1189">O2 on-exercise mean response time (MRT) and the gas exchange threshold (GET). The &#916;<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1190">O2/&#916;WR slope was calculated up to GET (S1), above GET (S2) and over the entire linear portion of the response (S T). Knee muscle endurance was measured by isokinetic dynamometry. As expected, peak <img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1191">O2 and muscle performance were lower in patients than controls (P < 0.05). Patients had significantly lower &#916;<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1192">O2/&#916;WR than controls, especially the S2 component (6.8 ± 1.5 vs 10.3 ± 0.6 mL·min-1·W-1, respectively; P < 0.001). There were significant relationships between &#916;<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1193">O2/&#916;WR (S T) and muscle endurance, MRT-<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1194">O2, GET and peak <img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1195">O2 in MM patients (P < 0.05). In fact, all patients with &#916;<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1196">O2/&#916;WR below 8 mL·min-1·W-1 had severely reduced peak <img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1197">O2 values (<60% predicted). Moreover, patients with higher cardiopulmonary stresses during exercise (e.g., higher &#916; ventilation/carbon dioxide output and &#916; heart rate/&#916;<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1198">O2) had lower &#916;<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1199">O2/&#916;WR (P < 0.05). In conclusion, a readily available, effort-independent index of aerobic dysfunction during dynamic exercise (&#916;<img border=0 width=11 height=13 src="548_files/image001.gif" v:shapes="_x0000_i1200">O2/&#916;WR) is typically reduced in patients with MM, being related to increased functional impairment and higher cardiopulmonary stress.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9%) for 15 days. Thyroidectomy increased OPG (~40%) and OPN (~75%) mRNA expression, while T3 treatment reduced OPG (~40%) and OPN (~75%) in Tx, and both (~50%) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

Stability of relative oxygen pulse curve during repeated maximal cardiopulmonary testing in professional soccer players

During cardiopulmonary exercise testing (CPET), stroke volume can be indirectly assessed by O2 pulse profile. However, for a valid interpretation, the stability of this variable over time should be known. The objective was to analyze the stability of the O2 pulse curve relative to body mass in elite athletes. VO2, heart rate (HR), and relative O2 pulse were compared at every 10% of the running time in two maximal CPETs, from 2005 to 2010, of 49 soccer players. Maximal values of VO2 (63.4 ± 0.9 vs 63.5 ± 0.9 mL O2&#8226;kg-1&#8226;min-1), HR (190 ± 1 vs188 ± 1 bpm) and relative O2 pulse (32.9 ± 0.6 vs 32.6 ± 0.6 mL O2&#8226;beat-1&#8226;kg-1) were similar for the two CPETs (P &gt; 0.05), while the final treadmill velocity increased from 18.5 ± 0.9 to 18.9 ± 1.0 km/h (P < 0.01). Relative O2 pulse increased linearly and similarly in both evaluations (r² = 0.64 and 0.63) up to 90% of the running time. Between 90 and 100% of the running time, the values were less stable, with up to 50% of the players showing a tendency to a plateau in the relative O2 pulse. In young healthy men in good to excellent aerobic condition, the morphology of the relative O2 pulse curve is consistent up to close to the peak effort for a CPET repeated within a 1-year period. No increase in relative O2pulse at peak effort could represent a physiologic stroke volume limitation in these athletes.

Immunological and biochemical parameters of patients with metabolic syndrome and the participation of oxidative and nitroactive stress

Metabolic syndrome (MS) is a multifactorial disease involving inflammatory activity and endothelial dysfunction. The aim of the present study was to evaluate the relationship between the changes in lipoperoxidation, in immunological and biochemical parameters and nitric oxide metabolite (NOx) levels in MS patients. Fifty patients with MS (4 males/46 females) and 50 controls (3 males/47 females) were studied. Compared to control (Mann-Whitney test), MS patients presented higher serum levels (P < 0.05) of fibrinogen: 314 (185-489) vs 262 (188-314) mg/dL, C-reactive protein (CRP): 7.80 (1.10-46.50) vs 0.70 (0.16-5.20) mg/dL, interleukin-6: 3.96 (3.04-28.18) vs 3.33 (2.55-9.63) pg/mL, uric acid: 5.45 (3.15-9.65) vs 3.81 (2.70-5.90) mg/dL, and hydroperoxides: 20,689 (19,076-67,182) vs 18,636 (15,926-19,731) cpm. In contrast, they presented lower (P < 0.05) adiponectin: 7.11 (3.19-18.22) vs 12.31 (9.11-27.27) µg/mL, and NOx levels: 5.69 (2.36-8.18) vs 6.72 (5.14-12.43) µM. NOx was inversely associated (Spearman&#8217;s rank correlation) with body mass index (r = -0.2858, P = 0.0191), insulin resistance determined by the homeostasis model assessment (r = -0.2530, P = 0.0315), CRP (r = -0.2843, P = 0.0171) and fibrinogen (r = -0.2464, P = 0.0413), and positively correlated with hydroperoxides (r = 0.2506, P = 0.0408). In conclusion, NOx levels are associated with obesity, insulin resistance, oxidative stress, and inflammatory markers. The high uric acid levels together with reactive oxygen species generation may be responsible for the reduced NO levels, which in turn lead to endothelial dysfunction. The elevated plasma chemiluminescence reflecting both increased plasma oxidation and reduced antioxidant capacity may play a role in the MS mechanism.

Correlation between total nitrite/nitrate concentrations and monoamine oxidase (types A and B) and semicarbazide-sensitive amine oxidase enzymatic activities in human mesenteric arteries from non-diabetic and type 2 diabetic patients

The aim of this study was to determine the correlation between total nitrite/nitrate concentrations (NOx) and the kinetic parameters of monoamine oxidase enzymes (MAO-A and MAO-B) and semicarbazide-sensitive amine oxidase (SSAO) in human mesenteric arteries. Arteries were from non-diabetic and type 2 diabetic patients with sigmoid or rectum carcinoma for whom surgery was the first option and who were not exposed to neo-adjuvant therapy. Segments of human inferior mesenteric arteries from non-diabetic (61.1 ± 8.9 years old, 7 males and 5 females, N = 12) and type 2 diabetic patients (65.8 ± 6.2 years old, 8 males and 4 females, N = 12) were used to determine NOx concentrations and the kinetic parameters of MAO-A, MAO-B and SSAO by the Griess reaction and by radiochemical assay, respectively. The NOx concentrations in arteries from diabetic patients did not differ significantly from those of the non-diabetic group (10.28 ± 4.61 vs 10.71 ± 4.32 nmol/mg protein, respectively). In the non-diabetic group, there was a positive correlation between NOx concentrations and MAO-B parameters: Km (r = 0.612, P = 0.034) and Vmax (r = 0.593, P = 0.042), and a negative correlation with the SSAO parameters: Km (r = -0.625, P = 0.029) and Vmax (r = -0.754, P = 0.005). However, in the diabetic group no correlation was found between NOx concentrations and the three kinetic parameters of the enzymes. These results suggest an important function of sympathetic nerves and vascular NOx concentrations in arteries of non-diabetic patients. Thus, these results confirm the importance of a balance between oxidants and antioxidants in the maintenance of vascular homeostasis to prevent oxidative stress.

Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI), and severe HI groups (N = 10 in each group at each time) on postnatal day 7 (P7) to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH) in the substantia nigra (SN). The mild and severe HI groups were exposed to hypoxia (8% O2/92% N2) for 90 and 150 min, respectively. The elevated plus-maze (EPM) test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT) and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold) and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05). The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2%) and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05). The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group) with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.

Cardiopulmonary exercise testing variables as predictors of long-term outcome in thoracic sarcoidosis

Cardiopulmonary exercise testing (CPET) plays an important role in the assessment of functional capacity in patients with interstitial lung disease. The aim of this study was to identify CPET measures that might be helpful in predicting the vital capacity and diffusion capacity outcomes of patients with thoracic sarcoidosis. A longitudinal study was conducted on 42 nonsmoking patients with thoracic sarcoidosis (median age = 46.5 years, 22 females). At the first evaluation, spirometry, the measurement of single-breath carbon monoxide diffusing capacity (D LCOsb) and CPET were performed. Five years later, the patients underwent a second evaluation consisting of spirometry and D LCOsb measurement. After 5 years, forced vital capacity (FVC)% and D LCOsb% had decreased significantly [95.5 (82-105) vs 87.5 (58-103) and 93.5 (79-103) vs 84.5 (44-102), respectively; P < 0.0001 for both]. In CPET, the peak oxygen uptake, maximum respiratory rate, breathing reserve, alveolar-arterial oxygen pressure gradient at peak exercise (P(A-a)O2), and &#916; SpO2 values showed a strong correlation with the relative differences for FVC% and D LCOsb% (P < 0.0001 for all). P(A-a)O2 &#8805;22 mmHg and breathing reserve &#8804;40% were identified as significant independent variables for the decline in pulmonary function. Patients with thoracic sarcoidosis showed a significant reduction in FVC% and D LCOsb% after 5 years of follow-up. These data show that the outcome measures of CPET are predictors of the decline of pulmonary function.

Influence of exercise modality on agreement between gas exchange and heart rate variability thresholds

The main purpose of this study was to investigate the level of agreement between the gas exchange threshold (GET) and heart rate variability threshold (HRVT) during maximal cardiopulmonary exercise testing (CPET) using three different exercise modalities. A further aim was to establish whether there was a 1:1 relationship between the percentage heart rate reserve (%HRR) and percentage oxygen uptake reserve (%V&#729;O2R) at intensities corresponding to GET and HRVT. Sixteen apparently healthy men 17 to 28 years of age performed three maximal CPETs (cycling, walking, and running). Mean heart rate and V&#729;O2 at GET and HRVT were 16 bpm (P<0.001) and 5.2 mL·kg-1·min-1 (P=0.001) higher in running than cycling, but no significant differences were observed between running and walking, or cycling and walking (P>0.05). There was a strong relationship between GET and HRVT, with R2 ranging from 0.69 to 0.90. A 1:1 relationship between %HRR and %V&#729;O2R was not observed at GET and HRVT. The %HRR was higher during cycling (GET mean difference=7%; HRVT mean difference=11%; both P<0.001), walking (GET mean difference=13%; HRVT mean difference=13%; both P<0.001), or running (GET mean difference=11%; HRVT mean difference=10%; both P<0.001). Therefore, using HRVT to prescribe aerobic exercise intensity appears to be valid. However, to assume a 1:1 relationship between %HRR and %V&#729;O2R at HRVT would probably result in overestimation of the energy expenditure during the bout of exercise.

The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O2&#8226;&#8722;) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

Use of the Wasserman equation in optimization of the duration of the power ramp in a cardiopulmonary exercise test: a study of Brazilian men

This study aimed to analyze the agreement between measurements of unloaded oxygen uptake and peak oxygen uptake based on equations proposed by Wasserman and on real measurements directly obtained with the ergospirometry system. We performed an incremental cardiopulmonary exercise test (CPET), which was applied to two groups of sedentary male subjects: one apparently healthy group (HG, n=12) and the other had stable coronary artery disease (n=16). The mean age in the HG was 47±4 years and that in the coronary artery disease group (CG) was 57±8 years. Both groups performed CPET on a cycle ergometer with a ramp-type protocol at an intensity that was calculated according to the Wasserman equation. In the HG, there was no significant difference between measurements predicted by the formula and real measurements obtained in CPET in the unloaded condition. However, at peak effort, a significant difference was observed between oxygen uptake (V&#729;O2)peak(predicted)and V&#729;O2peak(real)(nonparametric Wilcoxon test). In the CG, there was a significant difference of 116.26 mL/min between the predicted values by the formula and the real values obtained in the unloaded condition. A significant difference in peak effort was found, where V&#729;O2peak(real)was 40% lower than V&#729;O2peak(predicted)(nonparametric Wilcoxon test). There was no agreement between the real and predicted measurements as analyzed by Lin&#8217;s coefficient or the Bland and Altman model. The Wasserman formula does not appear to be appropriate for prediction of functional capacity of volunteers. Therefore, this formula cannot precisely predict the increase in power in incremental CPET on a cycle ergometer.

Acute but not chronic metabolic acidosis potentiates the acetylcholine-induced reduction in blood pressure: an endothelium-dependent effect

Metabolic acidosis has profound effects on vascular tone. This study investigated the in vivo effects of acute metabolic acidosis (AMA) and chronic metabolic acidosis (CMA) on hemodynamic parameters and endothelial function. CMA was induced by ad libitum intake of 1% NH4Cl for 7 days, and AMA was induced by a 3-h infusion of 6 M NH4Cl (1 mL/kg, diluted 1:10). Phenylephrine (Phe) and acetylcholine (Ach) dose-response curves were performed by venous infusion with simultaneous venous and arterial blood pressure monitoring. Plasma nitrite/nitrate (NOx) was measured by chemiluminescence. The CMA group had a blood pH of 7.15±0.03, which was associated with reduced bicarbonate (13.8±0.98 mmol/L) and no change in the partial pressure of arterial carbon dioxide (PaCO2). The AMA group had a pH of 7.20±0.01, which was associated with decreases in bicarbonate (10.8±0.54 mmol/L) and PaCO2 (47.8±2.54 to 23.2±0.74 mmHg) and accompanied by hyperventilation. Phe or ACh infusion did not affect arterial or venous blood pressure in the CMA group. However, the ACh infusion decreased the arterial blood pressure (&#916;BP: -28.0±2.35 mm Hg [AMA] to -4.5±2.89 mmHg [control]) in the AMA group. Plasma NOx was normal after CMA but increased after AMA (25.3±0.88 to 31.3±0.54 &#956;M). These results indicate that AMA, but not CMA, potentiated the Ach-induced decrease in blood pressure and led to an increase in plasma NOx, reinforcing the effect of pH imbalance on vascular tone and blood pressure control.

Inibição da síntese da ACC (ácido 1-carboxílico-1-aminociclopropano) oxidase em maçãs frigoconservadas em atmosfera controlada

Foi estudada a expressão da ACC oxidase em maçãs, cv. Jonagold, colhidas no estádio pré-climatérico e armazenadas sob refrigeração em atmosfera normal (0ºC, 95% UR - AN) e controlada (0ºC, 95% UR, 1,5% O2 e 2,5% CO2 - AC), durante 180 dias. Na instalação do experimento, aos 90 e aos 120 dias, foram coletadas amostras para a determinação da firmeza de polpa, da acidez total titulável, dos sólidos solúveis totais, da produção de etileno, da atividade ACC oxidase e para a detecção imunoquímica das isoformas desta enzima. A dosagem da atividade ACC oxidase foi realizada por cromatografia gasosa a partir de extrato protéico solúvel acrescido de 250µM de ACC, 10µL de sulfato ferroso e 30µL de ascorbato de sódio. Para a detecção imunoquímica utilizou-se a técnica "western blot", com anticorpos policlonais anti-ACC oxidase de maçã, após separação das proteínas em eletroforese e isoeletrofocalização. Não foi detectada ACC oxidase em maçãs pré-climatéricas. Porém, após 120 horas em condições ambientais, houve a síntese dessa enzima e um incremento na produção de etileno. A refrigeração não exerceu controle na síntese da ACC oxidase e produção de etileno, resultando em significativas perdas físico-químicas nas frutas armazenadas em AN. Já a utilização de AC permitiu controlar a via de biossíntese do etileno, pela inibição da síntese da ACC oxidase, mantendo o material com boa qualidade para o consumo in natura. A adição de ACC e dos cofatores aumentou a atividade ACC oxidase e alterou o pI da ACC oxidase.