Biological Research

This paper has two main purposes: A) to emphasize the role of the kidney in setting peripheral resistance, thus arterial blood pressure and flow distribution since birth to full grown; and B) to bring attention to the role that changes in pulse pressure and pulse velocity may have in the genesis of aging hypertension. A) According to the tonus regulation at basal steady conditions, the arterial system may be divided into three areas: I) the skin, in which the vascular tonus is regulated by heat; II) the kidney, whose vessels are regulated by glomerulo-tubular balance; and III) the rest of organs and tissues of the body, whose tonus is regulated according to the oxygen the cell needs to maintain its energetic equilibrium (ATP/ADP relationship). As area III has the higher flow and lowest equivalent resistance, the kidney is--hemodynamically--the organ that sets arterial blood pressure during life. Nevertheless, since birth to full grown, the kidney must progressively adjust the peripheral resistance of area III, in order to allow arterial blood pressure and renal distribution to match glomerular filtration with the increasing body metabolism. The tool that the kidney uses to adjust resistance of area III, thence arterial blood pressure and blood distribution, is the renin-angiotensin system. B) Aging decreases vascular distensibility. Lower distensibility of the arterial tree results in a progressive increase in amplitude and velocity of the pulse wave, then in its potency. Small resistance vessels must increase Bayliss response in order to reduce pulse impact on the precapillarial arteries. Structural changes in the resistance vessels, as well as in preglomerular arteries, should establish a feed-back mechanism responsible for the evolution of arterial blood pressure.
Histopathology in short-term experiments, series BLM + O 3 ; a) Lung inflammatory reaction (H-E x 80); b) Focal lung fibrosis (Masson’s trichromic stain x 80); c) Hemorrhagic bronchopneumonia (H-E x 80); d) Detachment of bronchiolar epithelium (H-E x 200). 
To study the effect of ozone in a chronically damaged lung, we used a bleomycin (BLM) induced pulmonary fibrosis model. Both endotracheal instillation of BLM and O3 exposure both produce lung inflammation and fibrosis. Oxidative stress would be a common mechanism of damage for both BLM and O3. Our aim was to assess lung injury induced by 5 and 60 days of intermittent exposure to 0.25 ppm O3 in rats with bleomycin-induced pulmonary fibrosis. Thirty-day-old Sprague Dawley rats were endotracheally instilled with BLM (1 U/100 g body weight) and, 30 days later, exposed to 0.25 ppm 03 (0.25 ppm 4 h per day, 5 days a week). Histopatology controls were instilled with saline and breathing room air. Histopathological evaluation of lungs was done 5 and 60 days after O3 exposure. BLM-induced lung damage did not change after 60 days of intermittent O3 exposure. Five days of O3 exposure increased the mean score of BLM-induced pulmonary inflammation and fibrosis (p=0.06). Frequency of bronchopneumonia increased from 1/7 to 6/6 (p <0.001), suggesting that a short-term exposure to O3 in a previously damaged lung might be a risk factor for developing further lung injury.
The effect of the medium composition and salinity on the carotenoid to chlorophyll “a” ratio in 28 day-old cultures of D. salina and D. bardawil , maintained at 25oC and 40 μ mol m -2 s -1 (n=3 ± SD). 
The effect of the medium composition and salinity on the α - and β -carotene percentage in 28 day- old cultures of D. salina strain CONC-007, maintained at 25oC and 40 μ mol m -2 s -1 (n=3 ± SD). 
The effect of the medium composition and salinity on the isomeric composition of β -carotene in 28 day-old cultures of D. salina and D. bardawil , maintained at 25oC and 40 μ mol m -2 s -1 (n=3 ± SD). 
Dunaliella salina and D. bardawil are well-known microalgae accumulating high levels of beta-carotene under growth-limiting conditions. In both taxa, this pigment is primarily composed of the isomers 9-cis and all-trans. The 9-cis beta-carotene occurs only in natural sources and is the most attractive from a commercial point of view. The conditions that enhance the preferred accumulation of 9-cis beta-carotene in D. salina are controversial and they have not been well established yet. This study examined the effect of salinity on the quantity and quality of total carotenoids and beta-carotene isomers accumulated by D. salina (strain CONC-007) and D. bardawil (strain ATCC 30861) grown in two media with different nutritional compositions (PES and ART) and at salt concentrations of 1M, 2M and 3M NaCl. Total carotenoids were determined by spectrophotometry and beta-carotene isomers, by HPLC. The highest carotenoid contents per cell were obtained at 2M NaCl in both taxa. In both media, an increase of the 9-cis/all-trans beta-carotene ratio was observed in D. bardawil when the salt concentration increased, with a maximum value of 2.6 (in ART medium at 3M NaCl). In D. salina this ratio did not exhibit the same pattern, and the salt concentrations for maximal ratios were different in both media. The highest ratio obtained for this strain was 4.3 (in ART medium at 2M NaCl).
Structure of the 6-Arylbenzimidazo [1,2-c]quinazoline derivatives
Effect of LPS, and the combination of PMA/LPS on TNF-a secretion and mRNA expression. HL-60 cells were stimulated either with LPS (10 μ g/mL) or PMA (1 μ M) + LPS (10 μ g/ mL) for 6 h and (A) mRNA expression level was determined through RT-PCR using GAPDH as an internal control and (B) TNF- α was quantified in a conditioned medium by means of ELISA. Figure 2A shows a representative RT-PCR of three independent experiments for TNF- α mRNA expression in response to, LPS or PMA+LPS (PL). Figure 2B shows the TNF- α secretory response of HL-60 cells exposed to, LPS or PMA+LPS (PL). Results in B are presented as means ± SEM of n= 5. ***p<0.001 vs control. 
Inhibition of PMA/LPS-mediated TNF- α production by 6-Arylbenzimidazo[1,2- c ] quinazoline derivatives. HL-60 cells were stimulated simultaneously for 6 h with PMA+LPS (PL) in the presence or absence of 100 μ M of the new 6-Arylbenzimidazo[1,2- c ]quinazoline derivatives. Pentoxyfilline (Px) was used at 100 μ M (Px100) and 2 mM (Px2) as a reference control. Each compound was added 30 min before stimulation and DMSO was used as a control. Results are presented as means ± SEM of n= 4. *p<0.001 vs PMA/LPS, **p<0.005 vs PMA/LPS. 
Effect of 6-Phenyl-benzimidazo[1,2- c ]quinazoline on TNF- α mRNA expression. HL-60 cells were stimulated simultaneously for 6 h with PMA/LPS (PL) in the presence or absence of 100 μ M of 6-Phenyl-benzimidazo[1,2- c ]quinazoline (G1). The compound G1 was added 30 min before stimulation and DMSO was used as a control. Figure 4A shows a representative RT-PCR of three independent experiments. Figure 4B shows the TNF- α mRNA expression, expressed as folds over the control. Results are presented as means ± SEM of n= 3. *p<0.001 vs PMA/LPS. C, control; PL, PMA/LPS. 
Cytotoxic effects of 6-Arylbenzimidazo[1,2- c ]quinazoline derivatives. HL-60 cells were incubated for 6 h in the presence or absence of 100 μ M of the new inhibitory 6- Arylbenzimidazo[1,2- c ]quinazoline derivatives and the LDH activity released into a conditioned medium was evaluated. Cytotoxicity is expressed as the % of LDH activity compared to the maximal released activity. Results are presented as means ± SEM of n= 3. No statistically differences versus control (DMSO) were observed. 
This study describes the effect of novel 6-Arylbenzimidazo[1,2-c]quinazoline derivatives as tumor necrosis factor alpha (TNF-alpha) production inhibitors. The newly synthesized compounds were tested for their in vitro ability to inhibit the lipolysaccharide (LPS) induced TNF-alpha secretion in the human promyelocytic cell line HL-60. The compound 6-Phenyl-benzimidazo[1,2-c]quinazoline, coded as Gl, resulted as the most potent inhibitor and with no significant cytotoxic activity. Thus, 6-Arylbenzimidazo[1,2-c]quinazoline derivatives may have a potential as anti-inflammatory agents.
Effect of protein extracts from inoculated and mocked-inoculated lemon seedlings on Alternaria alternata. 1a. Germination and development of mycelia of A. alternata . Conidia suspensions (10 6 conidia/mL) were subjected to treatments for 72 hours. Aliquots of 20 μ L of treated suspensions were placed into holes in APD contained in Petri dishes. These were incubated for 5 days at 28 ° C. Treatments at final concentrations: a) 10 mM sodium acetate pH 5.0; b) 200 μ g of commercial chitinase from S. marcescens (0.03 units/mg); c) 200 μ g total protein of homogenates from mocked inoculated lemon seedlings, and d) 200 μ g total protein of homogenates from inoculated lemon seedlings. Arrow indicates site of inoculation. 1b. Degradation of mycelia from A. alternata . The fungus was grown until the entire plate was covered by mycelia. 20 μ L of the following treatments were placed in holes. Final concentrations indicated: a) 10 mM sodium acetate pH 5.0; b) 200 μ g of commercial chitinase from S. marcescens (0.03 units/mg), c) 200 μ g total protein of homogenates from inoculated lemon seedlings, d) 200 μ g total protein of homogenates from mock-inoculated lemon seedlings and e) 200 μ g total protein of homogenates from inoculated lemon seedlings heated for 20 minutes at 100 ° C. 
Time course changes in total ß-1,3-glucanase activity in lemon seedlings after inoculation with A. alternata. ß-1,3glucanase maintained the time zero activity during the entire time period analyzed in lemon seedlings treated with aamanitin or with cycloheximide before fungal inoculation. Bars represent standard deviations.
In addition to phytoalexin synthesis, the defense response of intact Citrus limon seedlings against Alternaria alternata involves both constitutive and induced enzyme activities such as chitinases (Ch) and beta-1,3-glucanases (Glu). A alternata conidial germination was prevented by protein extracts from inoculated lemon seedlings, but also by extracts from mock-inoculated specimens. On the other hand, degradation of mycelia was accomplished only by protein extracts from inoculated seedlings. The presence of six Ch isoenzymes and of four Glu isoenzymes was detected in protein extracts from mock-inoculated seedlings. As a result of fungal inoculation, the isoenzyme pattern of Ch and Glu changed, making possible the detection of a new Ch isoenzyme and of three new Glu. Also, some constitutive Ch and Glu increased their enzyme activity, and those Ch that increased their activity also showed a broadening of their substrate specificity. These changes were prevented by alpha-amanitin and cycloheximide, suggesting that the presence of new Ch and Glu isoenzymes was due to de novo synthesis of proteins. Results suggest that constitutive Ch and Glu could act as pre-formed defense molecules in Citrus limon preventing A. alternata germination, while those induced after fungal inoculation of lemon seedlings could act along with the former, to produce lysis of fungal mycelia, resulting in a more efficient control of A. alternata development.
The biosynthetic pigment from Chromobacterium violaceum BB-78, 1,3-dihydro-2H-indol-2-one and its derivatives exhibit biological activities such as antimicrobial action, low hemolytic effects on red blood cells and in vitro trypanocide activity. A relatively high cytotoxicity on V-79 hamster fibroblast cells of the biosynthetic pigment was found, although with the methylol derivative the toxicity was almost eliminated. The methylol derivative exhibited similar toxicity as Nifurtimox, a known, commercial trypanocide compound.
Calcium release via intracellular Ca2+ release channels is a central event underpinning the generation of numerous, often divergent physiological processes. In electrically non-excitable cells, this Ca2+ release is brought about primarily through activation of inositol 1,4,5-trisphosphate receptors and typically takes the form of calcium oscillations. It is widely believed that information is carried in the temporal and spatial characteristics of these signals. Furthermore, stimulation of individual cells with different agonists can generate Ca2+ oscillations with dramatically different spatial and temporal characteristics. Thus, mechanisms must exist for the acute regulation of Ca2+ release such that agonist-specific Ca2+ signals can be generated. One such mechanism by which Ca2+ signals can be modulated is through simultaneous activation of multiple second messenger pathways. For example, activation of both the InsP3 and cAMP pathways leads to the modulation of Ca2+ release through protein kinase A mediated phosphoregulation of the InsP3R. Indeed, each InsP3R subtype is a potential substrate for PKA, although the functional consequences of this phosphorylation are not clear. This review will focus on recent advances in our understanding of phosphoregulation of InsP3R, as well as the functional consequences of this modulation in terms of eliciting specific cellular events.
Inositol 1,4,5-trisphosphate (InsP3) is an established calcium-mobilizing messenger, which is well-known to activate Ca2+ signaling in many cell types. Contractile cardiomyocytes express hormone receptors that are coupled to the production of InsP3. Such cardioactive hormones, including endothelin, may have profound inotropic and arrhythmogenic actions, but it is unclear whether InsP3 underlies any of these effects. We have examined the expression and localization of InsP3 receptors (InsP3Rs), and the potential role of InsP3 in modulating cardiac excitation-contraction coupling (EC coupling). Stimulation of electrically-paced atrial and ventricular myocytes with a membrane-permeant InsP3 ester was found to evoke an increase in the amplitudes of action potential-evoked Ca2+ transients and to cause pro-arrhythmic diastolic Ca2+ transients. All the effects of the InsP3 ester could be blocked using a membrane-permeant antagonist of InsP3Rs (2-aminoethoxydiphenyl borate; 2-APB). Furthermore, 2-APB blocked arrhythmias evoked by endothelin and delayed the onset of positive inotropic responses. Our data indicate that atrial and ventricular cardiomyocytes express functional InsP3Rs, and these channels have the potential to influence EC coupling.
The InsP3R Ca(2+)-release channel has biphasic dependence on cytoplasmic free Ca2+ concentration ([Ca2+]i). InsP3 activates gating primarily by reducing high [Ca2+]i inhibition. To determine whether relieving Ca2+ inhibition is sufficient for activation, we examined single-channels in low [Ca2+]i in the absence of InsP3 by patch clamping isolated Xenopus oocyte nuclei. For both endogenous Xenopus type 1 and recombinant rat type 3 InsP3R channels, spontaneous InsP3-independent activities with low open probability Po (approximately 0.03) were observed in [Ca2+]i < 5 nM, whereas none were observed in 25 nM Ca2+. These results establish the half-maximal inhibitory [Ca2+]i in the absence of InsP3 and demonstrate that the channel can be active when all of its ligand-binding sites are unoccupied. In the simplest allosteric model that fits all observations in nuclear patch-clamp studies, the tetrameric channel can adopt six conformations, the equilibria among which are controlled by two inhibitory, one activating Ca(2+)-binding, and one InsP3-binding sites in a manner similar to the Monod-Wyman-Changeux model. InsP3 binding activates gating by affecting the relative affinity for Ca2+ of one of the inhibitory sites in different channel conformations, transforming it into an activating site. Ca2+ inhibition of InsP3-liganded channels is mediated by an InsP3-independent second inhibitory site.
Expression analysis of TEX101 in various human tissues. Immunohistochemistry was performed to examine 28 types of tissues using a rabbit polyclonal antibody against TEX101. These organs covered a wide range, including cardiac muscle, lung, kidney, spleen, liver, esophagus, stomach, small intestine, colon, pancreas, salivary gland, ovary, uterine cervix, endometrium, breast, testis, prostate, hypophysis, thyroid gland, parathyroid gland, adrenal gland, thymus gland, tonsil, larynx, cerebellum, eye, peripheral nerves, and skin. The tissues sections were visualized using diaminobenzidine, and images were captured with a Leica DM2500 microscope (200×). Clear and strong staining was observed in the testis samples, but not in the other tissue samples, including ovary.
Expression characteristics of TEX101 in testis. The tissue localization of TEX101 in testis was investigated through immunohistochemistry. Strong staining of TEX101 was found in seminiferous tubules, but not in the connective tissue of testis (50×). In seminiferous tubules, TEX101 was highly expressed in spermatocytes and spermatids, but not in spermatogonia (400×).
Western blotting analysis of TEX101 in testis. TEX101 expression in human and mouse testis was examined by Western blotting. In human testis tissues, a single, specific band of 38 kDa was detected, while no band appeared at 21 kDa. In mouse testis, no band was detected under the same experimental conditions.
Expression analysis of TEX101 in testicular cancer. TEX101 expression in seminoma, yolk sac tumor, embryonal carcinoma, and normal testis tissue were examined using immunohistochemistry. The sections showed no staining of TEX101; however, all collected normal testis tissues possessed strong staining of TEX101, resulting in 100% positive expression.
Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.
The fact that Alzheimer's beta amyloid (Abeta) peptides forms cation channels in lipid bilayers was discovered during the course of our experiments in the laboratory of "Guayo" Rojas at NIH in Bethesda, Maryland (USA). Recently, we found that the Abeta ion channel could be blocked selectively with small peptides that copy the amino acid sequence of the predicted mouth region of the Abeta channel pore. We now have searched for the essential amino acid residues required for this blocking effect by mutations. We found that the ability of peptides to block Abeta channel activity could be lost by replacement of histidines 13 and 14 by alanine or lysine. The amino acid substitution also resulted in the loss of the capacity of the peptides to protect cells from Abeta cytotoxicity. These data thus contribute to the definition of the region of the Abeta sequence that participates in the formation of the channel pore. Additionally, these data support the hypothesis that the ion channel activity of Ab contributes significantly to the cytotoxic properties of Abeta. These data also emphasize the potential value in using inhibition of Abeta ion channel activity as an end point for Alzheimer's disease drug discovery.
I-V curve of whole cell Ca 2+ currents in normal and trisomic DRG neurons. Open squares represent normal cells (n=18); closed squares represent Ts16 cells (n=20). Points are means + S.E.M.  
Voltage-dependent activation of Ca 2+ currents. Values are normalized to peak current in each cell. Points are means + S.E.M. (n=18 and 20 for normal and trisomic cells, respectively). * Significantly different for p<.05.  
Voltage-dependent inactivation of Ca 2+ currents. Values are normalized to peak current in each cell. No significant difference was observed at any pre-pulse potential level between the normal and trisomic condition. Points are means + S.E.M.  
Down syndrome is determined by the presence of an extra copy of autosome 21 and is expressed by multiple abnormalities, with mental retardation being the most striking feature. The condition results in altered electrical membrane properties of fetal dorsal root ganglia (DRG) neurons, as in the trisomy 16 fetal mouse, an animal model of the human condition. Cultured trisomic DRG neurons from human and mouse fetuses present faster rates of depolarization and repolarization in the action potential compared to normal controls and a shorter spike duration. Also, trisomy 16 brain and spinal cord tissue exhibit reduced acetylcholine secretion. Therefore, we decided to study Ca2+ currents in cultured DRG neurons from trisomy 16 and age-matched control mice, using the whole-cell patch-clamp technique. Trisomic neurons exhibited a 62% reduction in Ca2+ current amplitude and reduced voltage dependence of current activation at -30 and -20 mV levels. Also, trisomic neurons showed slower activation kinetics for Ca2+ currents, with up to 80% increase in time constant values. Kinetics of the inactivation phase were similar in both conditions. The results indicate that murine trisomy 16 alter Ca2+ currents, which may contribute to impaired cell function, including neurotransmitter release. These abnormalities also may alter neural development.
Amyloid aggregation inhibitors reduce intracellular amyloid accumulation in CTb cells. CNh and CTb cells were differentiated for 5 days (see “Methods”) and were maintained for two additional days in culture in the absence or in the presence of different compounds (20 μ M) before staining with Congo Red (CR). Panels A and B: CNh (A) and CTb (B) cells stained with CR and observed under bright field microscopy. Arrows point to some examples of cells containing CR-positive vacuoles in both cell lines. Scale bar: 25 μ m. Panel C: Percentage of CR-positive cells in cultures treated with different compounds. Statistical significances were evaluated using unpaired Student’s t-test. * p < 0.02 compared to untreated CNh cells; **p < 0.02 compared to untreated CTb cells. 
Amyloid aggregation inhibitors are non-toxic to CNh and CTb cells. Viabilities, measured using the Live/Dead assay (as described in “Methods”), of CNh and CTb cultures treated with 2,4-DNP, 3-NP and 4-AN for 48 hours. Three independent assays were performed (2-4 cover slips per assay). Viabilities are expressed as percentage of live cells. The data correspond to means ± SEM of three independent assays. From three to seven thousand cells were counted in each experimental condition. No statistically significant differences in viabilities in comparison to control were found after the treatment with the small molecule inhibitors. 
Intracellular calcium concentrations in drug-treated cells. After 48 hours of incubation in the absence or in the presence of 20 μM 2,4-DNP, 3-NP or 4-AN, CTb or CNh cells were loaded with the fluorescent calcium indicator Indo-1. Recordings were obtained from 10-15 individual cells per cover slip (three cover slips per experiment) and were converted to intracellular calcium concentrations using a calibration curve, as described in "Methods". Data are means ± SEM of three independent experiments. Statistical significances were evaluated using unpaired Student's t-test. * p < 0.02 compared to the respective untreated control.
We have previously characterized a number of small molecule organic compounds that prevent the aggregation of the beta-amyloid peptide and its neurotoxicity in hippocampal neuronal cultures. We have now evaluated the effects of such compounds on amyloid precursor protein (APP) accumulation in the CTb immortalized cell line derived from the cerebral cortex of a trisomy 16 mouse, an animal model of Down's syndrome. Compared to a non-trisomic cortical cell line (CNh), CTb cells overexpress APP and exhibit slightly elevated resting intracellular Ca2+ levels ([Ca2+] inverted exclamation mark). Here, we show that the compounds 2,4-dinitrophenol, 3-nitrophenol and 4-anisidine decreased intracellular accumulation of APP in CTb cells. Those compounds were non-toxic to the cells, and slightly increased the basal [Ca2+] inverted exclamation mark. Results indicate that the compounds tested can be leads for the development of drugs to decrease intracellular vesicular accumulation of APP in trisomic cells.
Immunocytochemical localization of steroidogenic enzymes, cytochrome P450 side chain cleavage, 17-alpha-hydroxylase and aromatase, was performed in the ovaries of Scotophilus heathi during reproductive cycle, with reference to the period of delayed ovulation. Moderate immunoreactivity of side chain cleavage enzyme and 17-alpha-hydroxylase was observed mainly in thecal cells and interstitial cells of the ovarian stroma during quiescence. Thecal cells positive for 17-alpha-hydroxylase were found even around the primary follicles. The peak immunoreactivity for all the three enzymes was observed during recrudescence. It coincided with high circulating steroid levels during this period. In the stroma, immunoreactivity for side chain cleavage and 17-alpha-hydroxylase was so extensive that it almost occupied the entire interfollicular area of the ovary. Aromatase immunoreactivity declined, but side chain cleavage enzyme and 17-alpha-hydroxylase remained extensive during the period of delayed ovulation. This suggests a high androgen and low estrogen synthesis during the period of delayed ovulation. There was a marked decline in 17-alpha-hydroxylase and an increase in aromatase immunoreactivity during the preovulatory period, suggesting a decrease in androgen and increase in estrogen synthesis. The results suggest thecal cells and interstitial cells of the stroma as the major site of steroidogenesis in the ovary of S. heathi. Over production of androgen is attributed to the extensive development of 17-alpha-hydroxylase positive interstitial cells in the ovarian stroma, and this may be responsible for delayed ovulation in Scotophilus heathi.
Cardiomyocyte hypertrophy and left ventricle (LV) scar formation after MI. Top left and middle: Representative microphotographs of LV myocardium stained with H&E in Sham- operated (Sh-1 week) and infarcted rats (MI-1 week). Bottom left and middle : Representative photographs of hearts from Sham- operated (Sh-1 week) and infarcted rats (MI-1 week) at week 1 post MI stained with sirius red. MI-RR: Myocardial infarction- remote region, MI-IR: Myocardial infarction-infarcted region. 
Relative expression of IL-6, IL-23, TGF- β , IL-1 β and TNF- α mRNA after MI. (A to E): mRNA extracted from LV samples ( A to E ) from Sham (Sh) and infarcted rats (MI) was analyzed for the expression of cytokines by conventional PCR. Results are expressed as mean ±SEM, p<0.05. (*) p<0.05 MI-1 week vs Sh-1 week, (#) p<0.05 MI-4 weeks vs Sh-4 weeks and vs Sh-1 week. n=4 to 7 per group. 
mRNA expression of Th17-related cytokines in left ventricle (LV) and protein levels of IL-17A and IL-6 in LV and plasma, 1 week after MI. ( A and B): mRNA extracted from LV samples from Sham (Sh) and infarcted rats (MI) was analyzed for the expression of IL-17A, and IL-6 by real time PCR. (C to F): Total protein extracts (C and E) and plasma samples (D and F) from Sham-operated (Sh) and infarcted rats (MI) at week 1 post MI were analyzed by ELISA for IL-17A and IL-6 levels. MI-RR: samples from MI remote region, MI-IR: samples from MI infarcted region. Results are expressed as mean ±SEM. (*) p<0.05 vs Sh, (#) p<0.05 vs MI-RR. n=6 per group. 
IL-17A localization on the infarcted region of the left ventricle ( LV) by immunohistochemistry. Representative microphotographs of LV myocardium from infarcted (MI) rats at week 1 post MI. Left and middle: images of the infarcted region (IR) of the LV. As it is shown we found positive staining following an endothelial pattern in most areas of the infarction scar (left), with some small positive cells that were in the vicinity of blood vessels (arrows). Right: image of the remote non-infarcted region of the same group of animals (RR), cardiomyocytes can be seen without presence of positive staining on the remaining myocardium. 
Th17 cells, a recently described subtype of CD4+ effector lymphocytes, have been linked to cell-mediated autoimmune and inflammatory diseases as well as to cardiovascular diseases. However, the participation of IL-17A in myocardial ischemic injury has not been clearly defined. We therefore conducted the present study to evaluate IL-17A and Th17-related cytokine levels in a rat model of myocardial infarction (MI). MI was induced in male Sprague Dawley rats by coronary artery ligation. Controls were sham-operated (Sh) or non-operated (C). Blood and samples from the left ventricle (LV) were collected at weeks 1 and 4 post-MI. At week 1, MI animals exhibited increased IL-6, IL-23 and TGF-β mRNA levels with no apparent change in IL-17 mRNA or protein levels in whole LV. Only TGF-β mRNA remained elevated at week 4 post-MI. However, further analysis revealed that IL-17A mRNA and protein levels as well as IL-6 and IL-23 mRNA were indeed increased in the infarcted region, though not in the remote non infarcted region of the LV, except for IL-23 mRNA. The increased expression of IL-17A and Th17-related cytokines in the infarcted region of LV, suggests that this proinflammatory pathway might play a role in early stages of post MI cardiac remodelling.
DDRT products (HT11G-AP5 primer combination) in gel of polyacrylamide 6%, urea 8M, at 120V for 18h stained with silver nitrate. B: negative controls (without RNA). W: workers. IN: intercastes. Q: queens. The arrow indicates differential expression.
In colonies of Melipona scutellaris Latreille, 1811 workers can be found with four ganglion nerve cells, a morphological characteristic of the queen. It is hypothesized that these workers, called intercastes, or phenocopies, are phenotypically-like workers, but genotypically identical to queens due to this specific trait. Workers with the same number of ganglion as queens seem to be intercastes between queens and workers. Our objective was to analyze the mRNA pro files of workers, queens, and intercastes of M. scutellaris through DDRT-PCR. Three hundred (300) pupae with white eyes were collected and externally identified according to the number of abdominal nerve ganglions: workers (5 ganglions), queens (4 ganglions) and intercastes (4 ganglions). The analysis identified differentially expressed transcripts that were present only in workers, but absent in intercastes and queens, confirming the hypothesis, by demonstrating the environmental effect on the queen genotype that generated phenotype-like workers.
Alexander Lipschütz was born in Riga, Latvia, in 1883. He obtained his M.D. from the University of Göttingen, Germany, in 1901. He conducted research at the Universities of Zürich, Bonn, Göttingen, Bern and Vienna. He was full Professor of Physiology at the Universities of Dorpart (1919-1926), in Tartu, Estonia, and Concepción (1927-1936), in Chile. Later, he became the first Director of the Institute of Experimental Medicine, of the Chilean National Health Service. He authored 22 books and a large number of scientific papers, mostly on Endocrinology and Oncology. He directed 16 medical theses at the University of Concepción and 81 at the University of Chile. He was awarded with the first Chilean National Prize in Science (1969). He died in Santiago, Chile, in 1980.
Restriction enzyme analysis in a family (A) and an isolated case (B). Electrophoresis of restriction products run in 2,5% agarose and stained with SYBR Green. L, molecular weight ladder. Chromosome 7 regions studied for each individual are represented as vertical lines. The number in each allele represents absence (1) or presence (2) of the restriction site for Taq I or Pst I in markers XV-2c (top panel) or KM.19 (bottom panel), respectively. XK haplotypes were labeled as described on Table 1. Panel A: Family #005, child with unknown CFTR mutations (“f”: father, “x”: affected and “m”: mother), with their constructed haplotypes at the bottom. Panel B: Patient #299, a Δ F508 homozygote, with the constructed haplotypes shown on the right. 
Cystic fibrosis (CF) is caused by mutations in the CFTR gene. More than 1600 mutations have been described, with frequencies that differ worldwide according to the ethnic origin of patients. A small group of mutations are recurrent on several populations. It has been shown that they each tend occur on specific chromosome 7 haplotypes, supporting the notion of a single origin for them. Less than 50% of mutations in Chilean patients have been identified to date. To indirectly assess the possible presence of a predominant founder mutation in the remaining unknown alleles, we evaluated 2 polymorphic markers, XV-2c and KM.19, tightly linked to the CFTR locus. The study was done in Chilean CF patients with unknown or deltaF508 (DeltaF508) CFTR mutations and their haplotypes were compared to affected family-based controls. DeltaF508 showed marked linkage disequilibrium with XV-2c/KM.19 haplotype B, with 90% of alleles on that haplotype. There was no difference in haplotype distribution between unknown mutations and normal controls. These results support a European origin for DeltaF508 alleles in Chilean patients, and make unlikely the presence of a predominant founder mutation in the so-far unknown alleles.
Enrique Egaña-Barahona. Born Santiago, Chile, 10 March 1912. Deceased Santiago, Chile, 23 November 1997. MD, University of Chile, 1936. Rockefeller Foundation Fellow at Harvard University Medical School, 1940-1944. Professor of Pathophysiology, 1963; Director, Institute of Experimental Medicine, Faculty of medicine, University of Chile. Author of a Textbook on General Pathophysiology (1963) and of many scientific articles in Chilean and American medical journals. Strong supporter of evidence-based medicine as well as of medical education by involving students in short research projects.
One of the most eminent neuroscientists recently passed away in Paris. Professor Francisco Varela was a scholar that approached science with a remarkably broad and integrative perspective, deeply contributing to a diversity of fields, from mathematics to epistemology, from immunology to neuroscience. He was strongly influenced by Buddhism and actively participated in unraveling the relationship between science and spirituality. This article introduces a special edition of Biological Research dedicated to the memory of this great man. It contains a collection of valuable contributions by various authors who collaborated with Varela at different moments of his outstanding scientific career. Their articles cover most of the fields in which he made contributions.
The purpose of this report is to examine the biological sciences in Chile and South America in bibliographic terms -the number of papers each nation published from 1981-1991 and the number of citations to them in the international research literature. The database consists of 34,600 biological science papers from Argentina, Brazil, Chile, and Venezuela in the 1981-1991 Science Citation Index files of the Institute for Scientific Information. Twelve specialty areas were selected to represent the biological sciences of special interest to Chile: animal sciences, biochemistry/biophysics, environmental sciences, experimental biology/medicine, immunology, microbiology/cell biology, molecular biology/genetics, neurosciences, pharmacology, physiology, plant sciences, and reproductive sciences. Data are reported on the number of papers in these fields, combined, by authors based in Chile and other South American nations. In addition, time-series trends in the impact (average citations per paper) of Chilean research relative to South America as a whole, overall and in each specialty, are presented and discussed.
Between November 1997 and April 1998, several human gastroenteritis cases were reported in Antofagasta, a city in the north of Chile. This outbreak was associated with the consumption of shellfish, and the etiologic agent responsible was identified as Vibrio parahaemolyticus. This was the first report of this bacterium causing an epidemic in Chile. V. parahaemolyticus was the only pathogenic bacterium isolated from patient stools and from shellfish samples. These isolates were analyzed by polymerase chain reaction (PCR) amplification of the pR72H gene, a species-specific sequence. Based on the pR72H gene amplification pattern, at least three different isolates of V. parahaemolyticus were found. Two isolates (named amplicons A and C) generated PCR products of approximately 400 bp and 340 bp respectively, while another type of isolate designated B, did not generate a PCR product, regardless of which method of DNA purification was used. Sequence analysis of the amplicons A and C shows that they have an 80 bp and 183 bp conserved region at the 5' end of the gene. However, both isolates have different sequences at their 3' terminus and are also different from the pR72H sequence originally reported. Using this PCR assay we demonstrated that these three isolates were found in clinical samples as well as in shellfish. The warm seawater caused by the climatological phenomena "El Niño" perhaps favored the geographic dispersion of the bacterium (bacterial bloom) occurring in Antofagasta that occurred during that time of year.
This work describes the involvement of TRPC proteins in capacitative calcium entry (CCE) induced by 1alpha,25-dihydroxy-vitamin-D3 [1alpha,25(OH)2D3] in chick skeletal muscle and in rat osteoblast-like cells (ROS 17/2.8) and the role of the vitamin D receptor (VDR) in this non-genomic rapid response mediated by the hormone. We propose that an endogenous TRPC3 protein mediates 1alpha,25(OH)2D3 modulation of CCE in these cells, which seems to implicate VDR-TRPC3 association and the participation of an INAD-like scaffold protein.
Molecular understanding of the mechanism of excitation-contraction (EC) coupling in skeletal muscle has been made possible by cultured myotube models lacking specific dihydropyridine receptor (DHPR) subunits and ryanodine receptor type 1 (RyR1) isoforms. Transient expression of missing cDNAs in mutant myotubes leads to a rapid recovery, within days, of various Ca2+ current and EC coupling phenotypes. These myotube models have thus permitted structure-function analysis of EC coupling domains present in the DHPR controlling the opening of RyR1. The purpose of this brief review is to highlight advances made by this laboratory towards understanding the contribution of domains present in alpha1S and beta1a subunits of the skeletal DHPR to EC coupling signaling. Our main contention is that domains of the alpha1S II-III loop are necessary but not sufficient to recapitulate skeletal-type EC coupling. Rather, the structural unit that controls the EC coupling signal appears to be the alpha1S/beta1a pair.
Expression of IP-10 and CXCR3 in lymphocyte populations obtained from healthy subjects, non-obese patients with asthma and obese patients with asthma Five x 10 5 CD4+ lymphocytes were obtained from human peripheral blood (a) non-obese and (b) obese patients with intermittent, mild persistent, moderate persistent and severe persistent asthma and healthy subjects. The cells were isolated by the method of Böyum and purifi ed to CD4 + T cells by the negative selection technique and were cultured for 24 h. They were stained with fl uorochromo-conjugated (FITC or PE) anti human IP-10 and CXCR3 monoclonal antibodies. 10,000 events were analyzed by fl ow cytometry. The quadrants were set with the isotype control (FITC or PE-conjugated mouse IgG). Bold histograms and numbers in the dot plots represent the mean ± SD of positively stained cells from at least six independent experiments. The statistical differences between the groups were calculated using the Mann-Whitney U test. p <0.05 was considered signifi cant. 
MIP1 α and CCR5 expression in lymphocytes from peripheral blood Five x 10 5 CD4+ lymphocytes were obtained from non-obese and obese patients with intermittent, mild persistent, moderate persistent 
Resistin and IL-6 production by lymphocyte cells obtained from healthy subjects, lean patients and obese patients with different levels of severity of asthma Cell culture supernatants were analyzed by ELISA to determine IL-6 and resistin production. The bars represent the average of three independent experiments in each group and the healthy subjects (control group). Quantitative data were expressed as normal distribution histograms with confi dence intervals of 95%. 
Asthma studies suggest that alteration in the inflammation pattern may be associated with the severity of asthma. The aim of this study was to compare in vitro the expression of chemokines, chemokine receptors and cytokine production from CD4+ T human lymphocytes of asthmatic, both obese and non-obese patients with different severity levels of asthma. Lymphocytes were labeled with monoclonal anti-human CXCR3/IP-10, MIP-1a/CCR5 antibodies and were analyzed by flow cytometry. Cell culture supernatants were used to measure production of interleukin IL-6 and resistin by ELISA. CXCR3/IP-10 expression increased in non-obese patients with mild persistent asthma (2.2%, p<0.05), moderate persistent asthma (3%, p<0.003) and severe persistent asthma (4%, p<0.004); this effect was stronger in obese patients with severe persistent asthma (35%, p<0.004). MIP-1 α / CCR5 increased in non-obese patients with intermittent asthma (0.65%, p<0.05) and severe asthma (1.4%, p<0.03); in obese patients, this expression was greater in intermittent asthma (8%, p<0.05) and severe persistent asthma (12%, p<0.04). Resistin production strongly increased in obese patients with intermittent (976 ng/ml) and severe persistent asthma (795 ng/ml). IL-6 increased in both lean and obese persons; however, the highest value was registered in the group of severe persistent obese asthmatics (992 pg/ml). Obesity per se increased the inflammatory profile of chemokines / cytokines secreted by cells of the blood, increasing the inflammatory status in asthmatic patients. Resistin showed characteristics of a pro-inflammatory cytokine mainly in severely obese asthmatics.
IL-1b increased the poliferation of RASFs dose-dependently and EACT inhibits IL-1b-induced proliferation of RASFs. The proliferation of RASFs was evaluated with CCK-8 kit after culture for 3 days with different concentration of IL-1b (0.1-10 ng/mL) (A). The proliferation of RASFs was also evaluated after culture for 2 days with CCK-8 kit with/without IL-1b (1.0 ng/mL) and EACT (100 ug/mL) (B). Results are Presented as mean ± SD, n = 3, *p<0.01 versus no IL1b and EACT, † p<0.05 versus IL-1b without EACT. 
EACT inhibited IL-1b-induced production of MMPs and COX-2 in RASFs. To evaluate the expression of MMP-1, 3, TIMP-1 and COX-1, 2 protein, immunoblotting was performed. RASFs were cultured for 2 days with/without IL-1b (1.0 ng/mL) and EACT (100 ug/mL). The RNA and protein were extracted from RASFs and analysed by RT-PCR (A) and Western blotting (B). IL-1b enhanced the expression of MMP1, 3, TIMP-1 and COX-2 protein compared with the results without IL-1 b. EACT inhibited the IL-1b-induced expression of MMP-1. 3 and COX-2 protein. Results are presented as mean ± SD. n = 3. *p < 0.05 or 
The objective of this study is to determine the effects of Ethyl acetate fraction from Cudrania tricuspidata (EACT) on the interleukin-1b (IL-1b)-induced proliferation of rheumatoid synovial fibroblasts (RASFs) and production of matrix metalloproteinases (MMPs), cyclooxygenase (COX) and prostaglandin E2 (PGE2) by RASFs. The proliferation of RASFs was evaluated with CCK-8 reagent in the presence of IL-1b with/without EACT. The expression of MMPs, TIMP-1, COXs, PGE2 and intracellular MAPK signalings, including p-ERK, p-p38, p-JNK and NF-kB were examined by immunoblotting or semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and ELISA in conditions as described above. EACT inhibits IL-1β-induced proliferation of RASFs and MMP-1, 3, COX-2 mRNA and protein expression, PGE2 production induced with IL-1b. EACT also inhibits the phosphorylation of ERK-1/2, p38, JNK and activation of NF-kB by IL-1b. These results suggest that EACT might be involved in synovial fibroblast proliferation and MMPs, COX-2, and PGE2 production, which are involved in joint destruction in rheumatoid arthritis (RA), indicating that this might be a new therapeutic modality for management of rheumatoid arthritis.
The enzyme pyruvate kinase of Leishmania mexicana amazonensis presents two forms with different kinetic properties and behavior for the heterotrophic activator fructose 2,6 bisphosphate. Pyruvate kinase 1, which is isolated as a tetramer, is inhibited by this metabolite. The second activity, Pyruvate kinase 2, is activated by fructose 2,6 bisphosphate, which promotes the monomer-tetramer conversion of this enzyme.
In higher vertebrates, from amphibians to humans, epidemial maturation is a conserved developmental process. Using adult epidemial tissue and an established keratinocyte cell line, the mouse Nkx-2.3 homeobox gene was demonstrated, for the first time, to be expressed in mouse epidermal keratinocytes. Under the normal culture condition, the spontaneous aggregation phenomenon, a common initiation step of ES cell differentiation, and the induction of mouse adult K1 keratin, a marker of mature epidermal keratinocytes, were both observed in vitro when the Xenopus Nkx-2.3 gene was stably transfected into a mouse pluripotent P19 EC cell line. The induction of mouse K1 keratin by using its Xenopus orthologous gene in the mouse P19 cell implies that Nkx-2.3 may play a conserved role in the epidermal maturation of the mouse, as it does in that of the frog (Ma, 2004). However, the CAT assay study on frog adult keratin promoter could not find the induction of adult keratin. This implies there might not be a direct activation of its promoter.
Macrophages with parasites in the cytoplasm Hematoxiline & Eosine 1000x.
Congenital Chagas disease acquired special importance in Chile after the certification of the control of Triatoma infestans and transmission by blood donors affected with Trypanosoma cruzi. In order to establish adequate protocols for intervention and control in infected mother-neonate pairs in endemic zones of Chagas disease, we present partial results (2005-2008) of a pilot project which is being carried out in the Province of Choapa, IV Region, Chile, whose objectives are: determine the current prevalence of the disease in pregnant women, estimate the incidence of vertical transmission of T. cruzi to newborns, determine the lineages of the parasite present in mothers who do and do not transmit the disease, determine the prevalence of Chagas disease in maternal grandmothers of neonates and study placental histopathology. Preliminary results indicated that in this study period, 3.7% of the women who gave birth in the Province have Chagas disease and 2.5% of their newborns were infected. The most frequent T. cruzi genotypes found in mothers studied during pregnancy were TCI and TCIId, either alone or in mixed infections. A high percentage (74.3%) of the grandmothers studied was infected with the parasite. In 29 placentas from mothers with Chagas disease we observed edema, necrosis, fibrinoid deposits and slight lymphoplasmocyte infiltration. In three placentas we found erythroblastosis and in one of them amastigote forms of T. cruzi; this was one of the cases of congenital infection. The evaluation of the diagnostic and control protocols generated will allow us to determine if it has been possible to modify the natural history of vertical transmission of T. cruzi in Chile.
Mn2+ binding to PEP carboxykinases. The number of moles of Mn2+ bound per mole of enzyme monomer (ν,) is calculated from the signal intensity of the lowest-field transition and plotted against free Mn 2+ concentration. Wild-type () and Tyr207Leu () enzymes. The lines show the best fit of the data to equation (1). 
The functional significance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in K(m)CO₂, a 32-fold decrease in V(max), and a 6-fold decrease in K(m) for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO₂ and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.
Microdeletion 22q11 in humans causes velocardiofacial and DiGeorge syndromes. Most patients share a common 3Mb deletion, but the clinical manifestations are very heterogeneous. Congenital heart disease is present in 50-80% of patients and is a significant cause of morbidity and mortality. The phenotypic variability suggests the presence of modifiers. Polymorphisms in the VEGFA gene, coding for the vascular endothelial growth factor A, have been associated with non-syndromic congenital heart disease, as well as with the presence of cardiovascular anomalies in patients with microdeletion 22q11. We evaluated the association of VEGFA polymorphisms c.-2578C>A (rs699947), c.-1154G>A (rs1570360) and c.-634C>G (rs2010963) with congenital heart disease in Chilean patients with microdeletion 22q11. The study was performed using case-control and family-based association designs. We evaluated 122 patients with microdeletion 22q11 and known anatomy of the heart and great vessels, and their parents. Half the patients had congenital heart disease. We obtained no evidence of association by either method of analysis. Our results provide further evidence of the incomplete penetrance of the cardiovascular phenotype of microdeletion 22ql 1, but do not support association between VEGFA promoter polymorphisms and the presence of congenital heart disease in Chilean patients with this syndrome.
The Expression of GLUT1 in Human Cancer Association refers to an association between Glut1 with metastasis and or poor prognosis of the cancer. NR refers to data Not Reported by the authors. Expression refers to the level Glut1 in relation to relevant non-cancerous tissue. 
Regulators and signaling pathways involved in glucose transport. 
Mammalian cells depend on glucose as a major substrate for energy production. Glucose is transported into the cell via facilitative glucose transporters (GLUT) present in all cell types. Many GLUT isoforms have been described and their expression is cell-specific and subject to hormonal and environmental control. The kinetic properties and substrate specificities of the different isoforms are specifically suited to the energy requirements of the particular cell types. Due to the ubiquitousness of these transporters, their differential expression is involved in various disease states such as diabetes, ischemia and cancer. The majority of cancers and isolated cancer cell lines over-express the GLUT family members which are present in the respective tissue of origin under non-cancerous conditions. Moreover, due to the requirement of energy to feed uncontrolled proliferation, cancer cells often express GLUTs which under normal conditions would not be present in these tissues. This over-expression is predominantly associated with the likelihood of metastasis and hence poor patient prognosis. This article presents a review of the current literature on the regulation and expression of GLUT family members and has compiled clinical and research data on GLUT expression in human cancers and in isolated human cancer cell lines.
The present study shows for the first time the phenolic composition and the in vitro properties (antioxidant and inhibition of nitric oxide production) of Hypericum calabricum Sprengel collected in Italy. The content of hypericins (hypericin and pseudohypericin), hyperforin, flavonoids (rutin, hyperoside, isoquercetrin, quercitrin, quercetin and biapigenin) and chlorogenic acid of H. calabricum, have been determined. The ethyl acetate fraction from the aerial parts of H. calabricum exhibited activity against the radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 value of 1.6 jig/ml. The test for inhibition of nitric oxide (NO) production was performed using the murine monocytic macrophage cell line RAW 264.7. The ethyl acetate fraction had significant activity with an IC50 value of 102 jig/ml and this might indicate that it would have an anti-inflammatory effect in vivo.
Fingerprinting of the Cimicifuga foetida extract. HPLC-ELSD fi ngerprint chromatogram of CFE showing a typical C foetida profi le (top). LC-MS analysis confi rms this Asian species by detection of cimifugin and its glucoside (bottom). 
Cimicifuga foetida cytoxicity on MCF-7 cells. Sulforhodamine assay for MCF-7 cells grown in cell culture media containing tamoxifen (Panel A) or Cimicifuga foetida extract (Panel B) for different time periods. Each dot represents the difference between the absorbance registered for cells grown under experimental conditions and control conditions for an individual replicate. 
RT-PCR for Hsp-27 and β 2 -microglobulin on MCF-7 cells. RT-PCR in MCF-7 cells grown on the following conditions. Channel 1: Control, Channel 2: CFE 2 μg/L, Channel 3: CFE 20 μg/L, Channel 4: CFE 200 μg/L, Channel 5: DNA ladder (100 bp), Channel 6: CFE 2000 μg/L, Channel 7: Tamoxifen 480 ng/mL, Channel 8: β Estradiol 315 pM. Panel A: amplicons for Hsp27 (400 bp) and Panel B: amplicons for β 2 -microglobulin (267 bp), used as constitutive control. 
Cimicifuga foetida, an Asian Cimicifuga species, has been employed as a cooling and detoxification agent in traditional Chinese medicine since ancient times. For this herb, two cycloartane triterpene glycosides isolated from the rhizomes have demonstrated cytotoxicity on rat tumor and human cancer cell lines. Since human Hsp27 is increased in various human cancers and exhibits cytoprotective activity that affects tumorigenesis and the susceptibility of tumours to cancer treatment, the purpose of this research was to study the expression of this protein in MCF-7 breast cancer cells. To accomplish this aim, MCF-7 cells were exposed to different concentrations of Cimicifuga foetida extract showing a reduction in cell number measured by the sulforhodamine assay. In addition, the expression of Hsp-27 mRNA detected by RT-PCR and Hsp-27 protein detected by immnofluorescence was present in all conditions, except when using the highest concentration of Cimicifuga foetida extract (2,000 jig /L). We conclude that Hsp 27 expression at 2,000 jig /L Cimicifuga foetida extract is diminished. This is the first report showing the Hsp-27 expression after exposure to Cimicifuga foetida extract in MCF-7 cells.
Eleven antigenic clones were isolated from a Trypanosoma cruzi cDNA expression library using a pool of Chagasic sera from Venezuelan patients. These clones were tested with 26 Chagasic sera and 11 sera from patients with other diseases. One clone (Clone 2) reacted with the majority of the Chagasic sera and did not react with the non-Chagasic sera. Restriction enzyme digestion of Clone 2 DNA showed that it contained an insert of 5.2 kb. In a Southern blot of a pulse field gel electrophoresis, Clone 2 hybridized to three T. cruzi chromosomal fragments. By Northern blot analyses, it was observed that the clone hybridizes to a major T. cruzi RNA band of 0.97 kb and to two minor bands of 4.8 and 6.3 kb. The expression of these three RNAs was higher in trypomastigotes than in epimastigotes or spheromastigotes. Specific antibodies isolated against the beta-galactosidase-Clone 2 fusion protein expressed in E. coli reacted with a 28 kilodalton protein in T. cruzi.
Inhibition of PP2A activity isolated from Mytilus chilensis by phycotoxins. Assays were carried out with 22 mM p­NPP as substrate, as described in Materials and Methods. Filled squares, inhibition by Okadaic acid, filled circles, inhibition by DTX1 and filled triangles, inhibition by Microcystine L­R. In the figure, at a value of V i / V 0 = 0.5, the K i for Microcistine L­R is obtained. The K i( s) for OA and DTX1 were obtained in a similar way. 
Protein phosphatases are involved in many cellular processes. One of the most abundant and best studied members of this class is protein phosphatase type-2A (PP2A). In this study, PP2A was purified from the mussel Mytilus chilensis. Using both SDS-PAGE and size exclusion gel filtration under denaturant conditions, it was confirmed that the PP2A fraction was essentially pure. The isolated enzyme is a heterodimer and the molecular estimated masses of the subunits are 62 and 28 kDa. The isolated PP2A fraction has a notably high p-NPP phosphatase activity, which is inhibited by NaCl. The hydrolytic p-NPP phosphatase activity is independent of the MgCl2 concentration. The time courses of the inhibition of the PP2A activity of p-NPP hydrolysis by increasing concentrations of three phycotoxins that are specific inhibitors of PP2A are shown. Inhibitions caused by Okadaic acid, dinophysistoxin-1 (DTX1, 35-methylokadiac acid) and Microcystine L-R are dose-dependent with inhibition constants (Ki) of 1.68, 0.40 and 0.27 nM respectively. Microcystine L-R, the most potent phycotoxin inhibitor of PP2A isolated from Mytilus chilensis with an IC50 = 0.25 ng/ml, showed the highest specific inhibition effect an the p-NPP hydrolisis. The calculated IC50 for DTX1 and OA was 0.75 ng/ml and 1.8 ng/ml respectively.
(A) LPS profi les from S. fl exneri wild type and its isogenic O-antigen mutants. LPS samples from equal numbers of bacterial cells were loaded in each lane and were analyzed by Tricine/ SDS-polyacrylamide gel electrophoresis on a 12% acrylamide gel followed by silver staining. (B) Adhesion and (C) invasion of S. fl exneri wild type and mutants to polarized Caco-2 cells. The assays were performed in triplicate on at least three independent occasions. Averages ± standard errors (error bars) are shown. Values that are signifi cantly different from those of the wild type are indicated by * (p<0.05) or *** (p<0.001). The strains are 2457T (wt), MSF1107 ( Δ wzz pHS2 :: aph ), MSF1033 ( Δ wzzB ), MSF1749 ( Δ wzy ), MSF1210 ( Δ waaL ), MSF252 ( Δ mxiH :: aph ). 
Secretion of Ipa proteins by S. fl exneri wild type and mutants. Secretion was induced by incubating bacteria with Congo red (CR). Proteins in culture supernatants were electrophoresed and silver stained. The positions of Ipa proteins are indicated. Numbers indicate positions and sizes (in kDa) of standard proteins. 
(A) LPS profiles from S. flexneri wild type and complemented O-antigen mutants (B) Invasion of polarized Caco-2 epithelial cells by S. fl exneri wild type and complemented O-antigen mutants. The assays were performed in triplicate on at least three independent occasions. Averages ± standard errors (error bars) are shown. Values were not signifi cantly different from those of the wild-type S. fl exneri. The strains are 2457T/pDA12 (wt/vector), MSF1107/pDAPF2 (Δwzz pHS2 ::aph/wzz pHS2 + ) and MSF1033/pDABF3 (ΔwzzB/wzzB + ).
Effect of pre-treatment of Caco-2 cells with purifi ed LPS on bacterial invasion. (A) LPS profi les of samples purifi ed from S. fl exneri wild type and its isogenic O-antigen mutants. (B) Competition assays. Caco-2 cells were pre-treated with 2 or 5 mg/ mL of LPS obtained from each indicated strain before the addition of wild-type bacteria. Invasion assays were performed in triplicate on at least three independent occasions. Averages ± standard errors (error bars) are shown. Values were not significantly different from that of non treated Caco-2 cells. 
Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexneri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz pHS2. Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.
CYP2E1 enzyme is related to nonalcoholic steatohepatitis (NASH) due to its ability for reactive oxygen species production, which can be influenced by polymorphisms in the gene. The aim of this study was to investigate hepatic levels, activity, and polymorphisms of the CYP2E1 gene to correlate it with clinical and histological features in 48 female obese NASH patients. Subjects were divided into three groups: (i) normal; (ii) steatosis; and (iii) steatohepatitis. CYP2E1 protein level was assayed in microsomes from liver biopsies, and in vivo chlorzoxazone hydroxylation was determined by HPLC. Genomic DNA was isolated for genotype analysis through PCR. The results showed that liver CYP2E1 content was significantly higher in the steatohepatitis (45%; p=0.024) and steatosis (22%; p=0.032) group compared with normal group. Chlorzoxazone hydroxylase activity showed significant enhancement in the steatohepatitis group (15%, p=0.027) compared with the normal group. c2 rare allele of RsallPstl polymorphisms but no C allele of Dral polymorphism was positively associated with CHZ hydroxylation, which in turn is correlated with liver CYP2E1 content (r=0.59; p=0.026). In conclusion, c2 allele is positively associated with liver injury in NASH. This allele may determine a higher transcriptional activity of the gene, with consequent enhancement in pro-oxidant activity of CYP2E1 thus affording liver toxicity.
The distribution of prostaglandin-E2 (PGE2) and prostaglandin-F2 alpha (PGF2 alpha) was studied in subcellular fractions isolated from homogenates of human atrial fresh tissue by differential centrifugation. Right and left atrial samples were excised from the same heart of six patients with mitral valve disease at the time of open heart surgery. The atrial fractions investigated were mitochondrial (8,500 g pellet), microsomal (100,000 g pellet) and cytosol soluble (100,000 g supernatant) fractions. After extraction of prostaglandins from the three atrial fractions and separation of PGE from PGF series by chromatography on silicic acid column, these prostaglandins were measured by radioimmunoassay. The results showed that PGE2 and PGF2 alpha were located mainly in the soluble cytosolic fraction of right and left atrial tissue (p < 0.001). Furthermore, the prostaglandins levels were higher in left than in right atria of these patients (p < 0.001). The relation between prostaglandins heart generation in response to elevated work load of mitral valve disease is discussed.
Microsomal NADPH-dependent superoxide radical (O 2.- ) production (A), superoxide dismutase (SOD) activity (B), and O 2.- production/SOD activity ratios (C) in the liver of control rats and animals subjected to γ -hexachlorocyclohexane (HCCH) or L-3,3',5-triiodothyronine (T ) administration. 3 Control rats for HCCH and T treatments exhibited comparable 3 
Microsomal activity of p-nitrophenol hydroxylation (A) and western blot analysis of cytochrome P4502E1 (B) in the liver of control rats and animals subjected to γ hexachlorocyclohexane (HCCH) or L-3,3',5-triiodothyronine (T ) administration. Representative blots shown in the upper 3 panel in B contain 30 μ g of microsomal protein from a different rat per lane. Data on the lower panel in B show the densitometric analysis of the immunoblots presented in the upper panel; for this purpose, data from both groups of control rats were set to unity, and values from HCCH- and T -treated 3 rats were normalized to respective control group. Values shown correspond to means ± SE for 4-10 animals per group in A and 3 rats per group in B. The significance of the differences between mean values (P< 0.05) was assessed by one-way ANOVA and the Newman-Keuls , test, and it is 
Liver microsomal cytochrome P4502E1-dependent p-nitrophenol (PNP) hydroxylation and expression of cytochrome P4502E1 were studied in rats subjected to gamma-hexachlorocyclohexane (HCCH) or L-3,3,5-triiodothyronine (T3) administration as a possible mechanism contributing to superoxide radical (O2.-) generation. HCCH treatment (a single dose of 40 mg/kg body wt) produced a 43% increase in the content of total cytochrome P450, whereas T3 (daily doses of 0.1 mg/kg body wt for two consecutive days) led to a 37% decrease. NADPH-dependent O2.- generation was elevated by HCCH and T3, expressed as either per mg of protein or per nmol of cytochrome P450, with a 135% enhancement in the O2.- production/superoxide dismutase (SOD) activity ratios being observed in both conditions. This was partly due to depression of SOD activity. Concomitantly, the molecular activity of NADPH-cytochrome p450 reductase was enhanced by 90 and 69% by HCCH and T3, respectively. In these conditions, microsomal PNP hydroxylation showed increases of 58 and 45% in HCCH- and T3-treated rats over control values, respectively, with a parallel 31% (HCCH) and 41% (T3) enhancement in the content of cytochrome P4502E1 assessed by western immunoblotting. We conclude that HCCH and T3 enhance the expression and activity of cytochrome P4502E1 and that of NADPH-cytochrome P450 reductase in rat liver, regardless of the changes in total cytochrome P450 content, representing major contributory mechanisms to microsomal NADPH-dependent O2.- generation.
Several single-nucleotide polymorphisms have been identified in the human TNF gene promoter. The polymorphism at position-308 (TNF-308), which involves substituting G for A and designing the TNF2 allele, leads to a higher rate of TNF gene transcription than the wild-type TNF1 allele in in vitro expression studies. It has also been linked to increased susceptibility to a variety of illnesses. Using PCR-RFLP analysis we detected significant differences in the TNF-308 genotypes of Chilean and other populations. We conclude that there is a gradient in the distribution of the TNF2 allele according to ethnicity; we have also hypothesized that populations bearing a higher proportion of the TNF2 allele may have an increased predisposition toward or incidence of several chronic metabolic, degenerative, inflammatory and autoimmune diseases.
Growth curves for mycoplasma free or infected cells. Mycoplasma hyorhinis (Mhy) or Mycoplasma fermentans (Mf) infected 32D ( A ), AGS ( B ), COS-7 ( C ) and HeLa ( D ) cells. Control cells mean mycoplasma free cells. The assays were performed in triplicate and experiments were repeated on two separate occasions. Error bars represent SD. An asterisk (*) indicates a signifi cant difference at P< 0.05, compared to control cells. Double asterisks (**) indicate P< 0.01, compared to control cells. 
Apoptosis analysis of mycoplasma-infected 32D cells. Hoechst 33258 staining: Mycoplasma free 32D cells (Control- 32D), M. fermentas -infected 32D cells (Mf-32D) and M. hyorhinis infected 32D cells (Mhy-32D) were harvested and stained with 2 μg/ml Hoechst 33258 in serum free RPMI 1640 for 10-15 minutes at 37 ( A ). DNA ladder analysis: The full length DNAs were extracted from mycoplasmas infected and control 32D cells and separated on a 1.8% agarose gel. λ Hin d III/ Eco R I DNA ladder was used as DNA size marker ( B ). Protein analysis. 50 μg total proteins were extracted from mycoplasmas infected and control 32D cells, separated by SDS-PAGE and detected by enhanced chemiluminescence. Western blots were performed to detect the expression of genes related to apoptosis. β -actin was used as an internal reference ( C ). The assays were performed in three separate experiments. 
Hierarchical clustering analysis of gene expression patterns based on expression data. Normalized expression values for the microarrays were clustered with microarray algorithm SamCluster program. Vertical lines refer to cDNA microarray chips numbered First, Second, Third, Fourth, Fifth, Sixth, Seventh and Eighth, and each horizontal line refers to a gene. The colors represent the logarithmic intensity of the expressed genes. Red and green colors denote up-and down-regulated genes, respectively.
Identifi cation of mycoplasma-infection-specifi c genes related to proliferation, apoptosis and tumorigenesis with Northern blots. Total RNAs were extracted from mycoplasma-free 32D cells (Control-32D), Mycoplasma fermentans -infected 32D cells (Mf-32D) and Mycoplasma hyorhinis -infected 32D cells (Mhy- 32D). GAPDH, 28S rRNA and 18S rRNA were used as internal RNA loading controls. The assays were performed in duplicate and experiments were repeated twice. 
Infection of mycoplasmas has been linked to various human diseases including arthritis, pneumonia, infertility and cancer. While Mycoplasma hyorhinis and Mycoplasma fermentans have been detected in gastric adenocarcinomas, the mechanisms underlyine the pathogenesis are unknown. In this study, cell growth kinetics, Hoechst 33258 staining, DNA ladder assays, Western blotting analysis and cDNA microarray assays were performed to investigate the roles of M. hyorhinis and M. fermentans during infection of mammalian cells. Our data demonstrated that these mycoplasmas inhibid the growth of immortalised cell lines (32D and COS-7) ane tumor cell lines (HeLa and AGS). In addition, the infection of the 32D cell line with M. hyorhinis and M. fermentans induced compression of the nucleus, degradation of the cell genome and dysregulation of the expression of genes related to proliferation, apoptosis, tumorigenesis, signaling pathway and metabolism. Apoptosis related proteins Bcl-2, Bid and p53 were down-regulated, Fas was up-regulated and Bax was dysregulated in mycoplasma-infected 32D cells. Together, our data demonstrated that infection of mycoplasmas inhibitd cele growts through modification of gene expression profiles and post-translation modification of proliferation and apoptosis related proteins.
Hearing loss is the most common inherited sensorial deficiency in humans; about 1 in 1000 children suffer from severe or profound hearing loss at birth. Mutations in the GJB2 gene are the most common cause of prelingual, non-syndromic autosomal recessive deafness in many populations; the c.35delG mutation is the most common in Caucasian populations. The frequency of the c.35delG mutation was estimated in two samples of deaf patients from Santiago, Chile. Unrelated non-syndromic sensorioneural deaf patients were examined: Group 1 consisted of 47 unrelated individuals with neurosensory deafness referred to the Chilean Cochlear Implant Program; Group 2 included 66 school children with prelingual deafness attending special education institutions for deaf people. Individuals with profound to moderate isolated neurosensory hearing loss with unknown etiology were included. The presence of the c.35delG mutation was evaluated by the allele-specific polymerase chain reaction method (PCR), and in some cases it was confirmed by direct DNA sequencing of the coding region of the GJB2 gene. Deaf relatives were present in 20.3% of the cases. We found 19.5% (22/113) patients with the c.35delG mutation, 6 of them homozygous; these rates are similar to frequencies found in other Latin American countries.
Top-cited authors
Gareth Owen
  • Pontificia Universidad Católica de Chile
Ines Urquiaga
  • Pontificia Universidad Católica de Chile
Alfonso Valenzuela
  • University of Chile
Francisco Aboitiz
  • Pontificia Universidad Católica de Chile
Chin-Yuan Hsu
  • Chang Gung University