Curcumin induces human HT-29 colon adenocarcinoma cell apoptosis by activating p53 and regulating apoptosis-related protein expression

Curcumin, a major yellow pigment and active component of turmeric, has multiple anti-cancer properties. However, its molecular targets and mechanisms of action on human colon adenocarcinoma cells are unknown. In the present study, we examined the effects of curcumin on the proliferation of human colon adenocarcinoma HT-29 cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method and confirmed the curcumin-induced apoptosis by morphology and DNA ladder formation. At the same time, p53, phospho-p53 (Ser15), and other apoptosis-related proteins such as Bax, Bcl-2, Bcl-xL, pro-caspase-3, and pro-caspase-9 were determined by Western blot analysis. The colon adenocarcinoma cells were treated with curcumin (0-75 µM) for 0-24 h. We observed that p53 was highly expressed in HT-29 cells and curcumin could up-regulate the serine phosphorylation of p53 in a time- and concentration-dependent manner. An increase in expression of the pro-apoptotic factor Bax and a decrease in expression of the anti-apoptotic factor Bcl-2 were also observed in a time-dependent manner after exposure of 50 µM curcumin, while the expression of the anti-apoptotic factor Bcl-xL was unchanged. Curcumin could also down-regulate the expression of pro-caspase-3 and pro-caspase-9 in a time-dependent manner. These data suggest a possible underlying molecular mechanism whereby curcumin could induce the apoptosis signaling pathway in human HT-29 colon adenocarcinoma cells by p53 activation and by the regulation of apoptosis-related proteins. This property of curcumin suggests that it could have a possible therapeutic potential in colon adenocarcinoma patients.

Comparison of anaerobic threshold determined by visual and mathematical methods in healthy women

Several methods are used to estimate anaerobic threshold (AT) during exercise. The aim of the present study was to compare AT obtained by a graphic visual method for the estimate of ventilatory and metabolic variables (gold standard), to a bi-segmental linear regression mathematical model of Hinkley's algorithm applied to heart rate (HR) and carbon dioxide output (VCO2) data. Thirteen young (24 ± 2.63 years old) and 16 postmenopausal (57 ± 4.79 years old) healthy and sedentary women were submitted to a continuous ergospirometric incremental test on an electromagnetic braking cycloergometer with 10 to 20 W/min increases until physical exhaustion. The ventilatory variables were recorded breath-to-breath and HR was obtained beat-to-beat over real time. Data were analyzed by the nonparametric Friedman test and Spearman correlation test with the level of significance set at 5%. Power output (W), HR (bpm), oxygen uptake (VO2; mL kg-1 min-1), VO2 (mL/min), VCO2 (mL/min), and minute ventilation (VE; L/min) data observed at the AT level were similar for both methods and groups studied (P > 0.05). The VO2 (mL kg-1 min-1) data showed significant correlation (P < 0.05) between the gold standard method and the mathematical model when applied to HR (r s = 0.75) and VCO2 (r s = 0.78) data for the subjects as a whole (N = 29). The proposed mathematical method for the detection of changes in response patterns of VCO2 and HR was adequate and promising for AT detection in young and middle-aged women, representing a semi-automatic, non-invasive and objective AT measurement.

Simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene using quenched-FRET real-time PCR

Hereditary hemochromatosis (HH) is a common autosomal disorder of iron metabolism mainly affecting Caucasian populations. Three recurrent disease-associated mutations have been detected in the hemochromatosis gene (HFE): C282Y, H63D, and S65C. Although HH phenotype has been associated with all three mutations, C282Y is considered the most relevant mutation responsible for hemochromatosis. Clinical complications of HH include cirrhosis of the liver, congestive cardiac failure and cardiac arrhythmias, endocrine pancreatic disease, which can be prevented by early diagnosis and treatment. Therefore, a reliable genotyping method is required for presymptomatic diagnosis. We describe the simultaneous detection of the C282Y, H63D and S65C mutations in the hemochromatosis gene by real-time PCR followed by melting curve analysis using fluorescence resonance energy transfer (FRET) probes. The acceptor fluorophore may be replaced by a quencher, increasing multiplex possibilities. Real-time PCR results were compared to the results of sequencing and conventional PCR followed by restriction digestion and detection by agarose gel electrophoresis (PCR-RFLP). Genotypes from 80 individuals obtained both by the conventional PCR-RFLP method and quenched-FRET real-time PCR were in full agreement. Sequencing also confirmed the results obtained by the new method, which proved to be an accurate, rapid and cost-effective diagnostic assay. Our findings demonstrate the usefulness of real-time PCR for the simultaneous detection of mutations in the HFE gene, which allows a reduction of a significant amount of time in sample processing compared to the PCR-RFLP method, eliminates the use of toxic reagents, reduces the risk of contamination in the laboratory, and enables full process automation.

Antioxidant and antimicrobial activities of bitter and sweet apricot (Prunus armeniaca L.) kernels

Abstract The present study describes the in vitro antimicrobial and antioxidant activity of methanol and water extracts of sweet and bitter apricot (Prunus armeniaca L.) kernels. The antioxidant properties of apricot kernels were evaluated by determining radical scavenging power, lipid peroxidation inhibition activity and total phenol content measured with a DPPH test, the thiocyanate method and the Folin method, respectively. In contrast to extracts of the bitter kernels, both the water and methanol extracts of sweet kernels have antioxidant potential. The highest percent inhibition of lipid peroxidation (69%) and total phenolic content (7.9 ± 0.2 µg/mL) were detected in the methanol extract of sweet kernels (Hasanbey) and in the water extract of the same cultivar, respectively. The antimicrobial activities of the above extracts were also tested against human pathogenic microorganisms using a disc-diffusion method, and the minimal inhibitory concentration (MIC) values of each active extract were determined. The most effective antibacterial activity was observed in the methanol and water extracts of bitter kernels and in the methanol extract of sweet kernels against the Gram-positive bacteria Staphylococcus aureus. Additionally, the methanol extracts of the bitter kernels were very potent against the Gram-negative bacteria Escherichia coli (0.312 mg/mL MIC value). Significant anti-candida activity was also observed with the methanol extract of bitter apricot kernels against Candida albicans, consisting of a 14 mm in diameter of inhibition zone and a 0.625 mg/mL MIC value.

Association of HDL cholesterol and triglycerides with mortality in patients with heart failure

It has been demonstrated that there is an association between serum lipoproteins and survival rate in patients with ischemic cardiomyopathy, as well as in patients with non-ischemic causes of heart failure. We tested the hypothesis of an association between serum lipoprotein levels and prognosis in a cohort of outpatients with heart failure, including Chagas' heart disease. The lipid profile of 833 outpatients with heart failure in functional classes III and IV of the New York Heart Association, with a mean age of 46.9 ± 10.6 years, 655 (78.6%) men and 178 (21.4%) women, was studied from April 1991 to June 2003. The survival rate was estimated by the Kaplan-Meyer's method and the Cox proportional hazards models. Etiology of heart failure was ischemic cardiomyopathy in 171 (21%) patients, Chagas' heart disease in 144 (17%), hypertensive cardiomyopathy in 136 (16%), and other etiologies in 83 (10%). In 299 (36%) patients, heart failure was ascribed to idiopathic dilated cardiomyopathy. Variables significantly associated with mortality were age (hazard ratio, HR = 1.02; 95%CI = 1.01-1.03; P = 0.0074), male gender (HR = 1.77; 95%CI = 1.2-2.62; P = 0.004), idiopathic dilated cardiomyopathy (HR = 1.81; 95%CI = 1.16-2.82; P = 0.0085), serum triglycerides (HR = 0.97; 95%CI = 0.96-0.98; P < 0.0001), and HDL cholesterol (HR = 0.99; 95%CI = 0.99-1.0; P = 0.0280). Therefore, higher serum HDL cholesterol and higher serum triglycerides were associated with lower mortality in this cohort of outpatients with heart failure.

Expression of E-cadherin, Snail and Hakai in epithelial cells isolated from the primary tumor and from peritumoral tissue of invasive ductal breast carcinomas

Epithelial intercellular cohesion, mainly mediated by E-cadherin (CDH1) expression and function, may be deregulated during cancer cell invasion of adjacent tissues and lymphatic and vascular channels. CDH1 expression is down-modulated in invasive lobular breast carcinomas but its regulation in invasive ductal carcinomas (IDC) is less clear. CDH1 expression is repressed by transcription factors such as Snail (SNAI1) and its product is degraded after Hakai ubiquitination. We compared CDH1, SNAI1 and HAKAI mRNA expression in IDC and paired adjacent normal breast tissue and evaluated its relation with node metastasis and circulating tumor cells. Matched tumor/peritumoral and blood samples were collected from 30 patients with early IDC. Epithelial cells from each compartment (tumor/peritumoral) were recovered by an immunomagnetic method and gene expression was determined by real time RT-PCR. There were no differences in CDH1, SNAI1 and HAKAI mRNA expression between tumor and corresponding peritumoral samples and no differential tumoral gene expression according to nodal involvement. Another 30 patients with a long-term follow-up (at least 5 years) and a differential prognosis (good or poor, as defined by breast cancer death) had E-cadherin and Snail protein detected by immunohistochemistry in tumor samples. In this group, E-cadherin-positive expression, but not Snail, may be associated with a better prognosis. This is the first report simultaneously analyzing CDH1, SNAI1 and HAKAI mRNA expression in matched tumor and peritumoral samples from patients with IDC. However, no clear pattern of their expression could distinguish the invasive tumor compartment from its adjacent normal tissue.

A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-β1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-β1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-β1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-β1. However, ED5 has no effect on VSMC apoptosis.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Reproducibility of heart rate variability parameters measured in healthy subjects at rest and after a postural change maneuver

Heart rate variability (HRV) provides important information about cardiac autonomic modulation. Since it is a noninvasive and inexpensive method, HRV has been used to evaluate several parameters of cardiovascular health. However, the internal reproducibility of this method has been challenged in some studies. Our aim was to determine the intra-individual reproducibility of HRV parameters in short-term recordings obtained in supine and orthostatic positions. Electrocardiographic (ECG) recordings were obtained from 30 healthy subjects (20-49 years, 14 men) using a digital apparatus (sampling ratio = 250 Hz). ECG was recorded for 10 min in the supine position and for 10 min in the orthostatic position. The procedure was repeated 2-3 h later. Time and frequency domain analyses were performed. Frequency domain included low (LF, 0.04-0.15 Hz) and high frequency (HF, 0.15-0.4 Hz) bands. Power spectral analysis was performed by the autoregressive method and model order was set at 16. Intra-subject agreement was assessed by linear regression analysis, test of difference in variances and limits of agreement. Most HRV measures (pNN50, RMSSD, LF, HF, and LF/HF ratio) were reproducible independent of body position. Better correlation indexes (r > 0.6) were obtained in the orthostatic position. Bland-Altman plots revealed that most values were inside the agreement limits, indicating concordance between measures. Only SDNN and NNv in the supine position were not reproducible. Our results showed reproducibility of HRV parameters when recorded in the same individual with a short time between two exams. The increased sympathetic activity occurring in the orthostatic position probably facilitates reproducibility of the HRV indexes.

Hydrogen sulfide postconditioning protects isolated rat hearts against ischemia and reperfusion injury mediated by the JAK2/STAT3 survival pathway

The JAK2/STAT3 signal pathway is an important component of survivor activating factor enhancement (SAFE) pathway. The objective of the present study was to determine whether the JAK2/STAT3 signaling pathway participates in hydrogen sulfide (H2S) postconditioning, protecting isolated rat hearts from ischemic-reperfusion injury. Male Sprague-Dawley rats (230-270 g) were divided into 6 groups (N = 14 per group): time-matched perfusion (Sham) group, ischemia/reperfusion (I/R) group, NaHS postconditioning group, NaHS with AG-490 group, AG-490 (5 µM) group, and dimethyl sulfoxide (DMSO; <0.2%) group. Langendorff-perfused rat hearts, with the exception of the Sham group, were subjected to 30 min of ischemia followed by 90 min of reperfusion after 20 min of equilibrium. Heart rate, left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of increase or decrease of left ventricular pressure (± dp/dt max) were recorded. Infarct size was determined using triphenyltetrazolium chloride (TTC) staining. Myocardial TUNEL staining was used as the in situ cell death detection method and the percentage of TUNEL-positive nuclei to all nuclei counted was used as the apoptotic index. The expression of STAT3, bcl-2 and bax was determined by Western blotting. After reperfusion, compared to the I/R group, H2S significantly improved functional recovery and decreased infarct size (23.3 ± 3.8 vs 41.2 ± 4.7%, P < 0.05) and apoptotic index (22.1 ± 3.6 vs 43.0 ± 4.8%, P < 0.05). However, H2S-mediated protection was abolished by AG-490, the JAK2 inhibitor. In conclusion, H2S postconditioning effectively protects isolated I/R rat hearts via activation of the JAK2/STAT3 signaling pathway.

A novel noninvasive method to detect rejection after heart transplantation

Prompt and accurate detection of rejection prior to pathological changes after organ transplantation is vital for monitoring rejections. Although biopsy remains the current gold standard for rejection diagnosis, it is an invasive method and cannot be repeated daily. Thus, noninvasive monitoring methods are needed. In this study, by introducing an IL-2 neutralizing monoclonal antibody (IL-2 N-mAb) and immunosuppressants into the culture with the presence of specific stimulators and activated lymphocytes, an activated lymphocyte-specific assay (ALSA) system was established to detect the specific activated lymphocytes. This assay demonstrated that the suppression in the ALSA test was closely related to the existence of specific activated lymphocytes. The ALSA test was applied to 47 heart graft recipients and the proliferation of activated lymphocytes from all rejection recipients proven by endomyocardial biopsies was found to be inhibited by spleen cells from the corresponding donors, suggesting that this suppression could reflect the existence of activated lymphocytes against donor antigens, and thus the rejection of a heart graft. The sensitivity of the ALSA test in these 47 heart graft recipients was 100%; however, the specificity was only 37.5%. It was also demonstrated that IL-2 N-mAb was indispensible, and the proper culture time courses and concentrations of stimulators were essential for the ALSA test. This preliminary study with 47 grafts revealed that the ALSA test was a promising noninvasive tool, which could be used in vitro to assist with the diagnosis of rejection post-heart transplantation.

Chemical characterization and antimicrobial activity of essential oils of salvia L. species

In this work, the essential oils of S. officinalis, S. sclarea, S. lavandulifolia and S. triloba were chemically analyzed by gas chromatography coupled to a mass spectrometry detector (GC/MSD), and their antimicrobial activity was tested against 10 microorganisms using the disk diffusion method and the Minimum Inhibitory Concentration (MIC) technique. The following major compounds were identified in the essential oils: α - and β-thujone, camphor and 1,8-cineole, except in S. sclarea, where linalool, linalyl acetate and α-terpineol were the major constituents. The antimicrobial activity showed significant differences (p < 0.05) only when obtained by the MIC method. Gram-positive microorganisms presented larger sensitivity for the essential oils. The lowest MIC was observed when Staphylococcus aureus was exposed to 2.31 mg.mL-1 of S. lavandulifolia essential oil, while the highest MIC value was obtained when Shigella flexneri was exposed to 9.25 mg.mL-1 of the same essential oil, thus demonstrating that this essential oil may be effective as a bacteriostatic agent against Gram-positive microorganisms.

Experimental results and modeling of poultry carcass cooling by water immersion

Poultry carcasses have to be chilled to reduce the central breast temperatures from approximately 40 to 4 °C, which is crucial to ensure safe products. This work investigated the cooling of poultry carcasses by water immersion. Poultry carcasses were taken directly from an industrial processing plant and cooled in a pilot chiller, which was built to investigate the influence of the method and the water stirring intensity on the carcasses cooling. A simplified empiric mathematical model was used to represent the experimental results. These results indicated clearly that the understanding and quantification of heat transfer between the carcass and the cooling water is crucial to improve processes and equipment. The proposed mathematical model is a useful tool to represent the dynamics of carcasses cooling, and it can be used to compare different chiller operational conditions in industrial plants. Therefore, this study reports data and a simple mathematical tool to handle an industrial problem with little information available in the literature.

Antioxidant activity, cito- and phototoxicity of pomegranate (Punica granatum L.) seed pulp extract

In the present work, a hydroethanolic extract was prepared from the entire seeds of pomegranate [Punica granatum L. (Punicaceae)] with Cachaça, a distilled Brazilian alcoholic beverage, protected from light for an 80-hour period. The desorption curve of the seeds, presented an optimal time extraction of approximately 24 hours. The extract was divided into two samples: protected from light, (Extract 1), or not, (Extract 2). The extracts were characterized by UV-Visible absorption spectroscopy, quantification of total phenolics by the Folin-Ciocalteu method, and the antioxidant activity was determined by the DPPH quenching method. Extract 2 presented 9.8% less total polyphenols than Extract 1. The pomegranate seeds extract lost 79% of its antioxidant activity during light exposure. Extract 1 up to 3% (w/v) showed neither cyto nor phototoxicity in the Hela cells. In conclusion, Punica granatum L. seeds contain a significant total polyphenol and TEAC amount and they can be used in simple extractive process, by direct contact with Cachaça in up to 80 hours in the darkness, which gives it good coloration, taste, and smell. This extract showed neither cytotoxicity nor post-irradiation phototoxicity with solar simulator even though the extract proved photoinstable.

Physical-chemical, caloric and sensory characterization of light jambolan (Syzygium cumini Lamarck) jelly

In Brazil, several little economically explored fruits have good potential as raw material for the agro-industry. This study aimed to produce and determine the physical-chemical and sensory characteristics of light jambolan jelly. This fruit has intense purple color, which gave the jellies - both standard and light - a quite attractive visual aspect. The light jellies exhibited similar physical-chemical characteristics to the ones developed through the conventional method and; with the proportion of sweeteners used, the caloric values of the formulations were reduced to the range of 41 to 53%, attending the requirements of the Brazilian legislation for this type of product. The sensory profile obtained for the 4 light formulations developed, clearly showed the tasters' preference for the jelly elaborated with the association of cyclamate and saccharin. Thus, the results revealed good perspectives for the application of this fruit in the food industry.