Scielo RSS <![CDATA[Biological Research]]> https://scielo.conicyt.cl/rss.php?pid=0716-976020090002&lang=en vol. 42 num. 2 lang. en <![CDATA[SciELO Logo]]> https://scielo.conicyt.cl/img/en/fbpelogp.gif https://scielo.conicyt.cl <![CDATA[<b>Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome <i>b<sub>5 </sub></i>reductase from <i>Mucor racemosus </i>PTCC 5305 in <i>E. coli</i></b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200001&lng=en&nrm=iso&tlng=en The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73%) with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli. <![CDATA[<b>Extraction of high-quality host DNA from feces and regurgitated seeds</b>: <b>a useful tool for vertebrate ecological studies</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200002&lng=en&nrm=iso&tlng=en DNA extraction methods for genotyping non-invasive samples have led to great advances in molecular research for ecological studies, and have been particularly useful for analyzing threatened species. However, scarce amounts of fragmented DNA and the presence of Taq polymerase inhibitors in non-invasive samples are potential problems for subsequent PCR amplifications. In this study we describe a novel technique for extracting DNA from alimentary tract cells found on external surfaces of feces and regurgitated seeds. The presence of contaminants and inhibitors is minimized and samples are preserved intact for use in other ecological research (e.g. trophic studies). The amplification efficiency and purity of the extracted DNA from feces were significantly higher than in commonly used extraction procedures. Moreover, DNA of two bird species was identified from seeds expelled by regurgitation. Therefore, this method may be suitable for future ecological studies of birds, and other vertebrate groups. <![CDATA[<b>The effect of crude ethanol extract and fractions of <i>Hyptidendron canum </i>(Pohl ex Benth.) Harley on the hepatopancreas of <i>Oreochromis niloticus </i>L</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200003&lng=en&nrm=iso&tlng=en Hyptidendron canum (Pohl ex Benth.) Harley is a native tree of the Brazilian Savannah. The fish Oreochromis niloticus L. was used as an experimental model to determine the bioactivity of the crude ethanol extract as well as ethyl acetate, hexanic and chloroform fractions obtained from its leaves. The plant ethanol extract and fractions were administered to the fish orally with their feed. Twenty four hours later, the fish were sacrificed and their livers dissected, fixed in neutral formalin, embedded in paraffin and sectioned. Histological analyses were performed using Masson's trichrome and Haematoxylin-Eosin. Histochemical studies were performed using Feulgen, PAS (Periodic Acid Schiff) and PAS + salivary amylase and Sudan IV stain. The qualitative analysis of the material showed that both the crude ethanol extract and the fractions from H. canum induced vasoactive activity, causing vasodilation and vascular congestion, and the hexanic fraction also caused an apparent proliferation of capillaries. Hepatopancreas toxicity was evident through inflammatory processes. Pancreatic (chloroform fraction) and hepatic alterations, hemorrhagic spots and necroses were observed in fish treated with-ethanol extract and fractions. This study is the first description of the biologic action of the crude ethanol extract and the hexane, ethyl acetate and chloroform fractions in fish. <![CDATA[<b>Organic and inorganic selenium compounds produce different protein patterns in the blood plasma of rats</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200004&lng=en&nrm=iso&tlng=en Some selenium compounds offer important health benefits when administered at supranutritional doses, such as improvement of the immune system and of male fertility, and the prevention of some types of cancer. The traditional selenium indexes do not account for the metabolic status of this element among replete individuals. As a consequence, there is a need for new indexes that distinguish between repletion statuses of selenium. The aim of this work was to indentify some plasmatic proteins that respond to supranutritional doses of selenium, which could be proposed as new protein markers of selenium intake. The effect on rats of dietary supplementation with either selenomethylselenocysteine (SMSeC) or sodium-selenate on some blood plasma proteins was investigated. Two experimental groups consisting of six rats each were fed a basic diet supplemented with either SMSeC or sodium-selenate at 1.9 mg-Se / g-diet for ten weeks. The control group was fed a diet that contained the recommended selenium dose (0.15 mg-Se / g-diet). The changes in the abundance of a group of plasmatic proteins were quantified and analysed statistically. Haptoglobin, apolipoprotein E and transthyretin increased their abundance after diet supplementation with either form of selenium. HNF6 was responsive only to SMSeC, whereas fibrinogen responded only to sodium-selenate. We postulate that the protein patterns observed in this work could be proposed as new molecular biology-based markers of selenium intake. <![CDATA[<b>Antioxidant Properties and Total Phenolic Content of Eight <i>Salvia </i>Species from Turkey</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200005&lng=en&nrm=iso&tlng=en Methanolic extracts of eight Salvia species, namely S. aethiopis, S. candidissima, S. limbata, S. microstegia, S. nemorosa, S. pachystachys, S. verticillata, and S. virgata, sampled from Eastern Anatolia in Turkey, were screened for their possible antioxidant activities by two complementary test systems, namely DPPH free radical scavenging and b-carotene/linoleic acid. Total phenolic content of the extracts of Salvia species were performed Folin-Ciocalteu reagent and gallic acid used as standard. A wide variation has been observed among species in terms of antioxidant activity and total phenolic content. In both DPPH and b-carotene system, the most active plant was Salvia verticillata with a value of IC50=18.3 μg/ml and 75.8%, respectively. This specie also has the highest total phenolic content (167.1 mgGAE/g DW). The total amount of phenolics was between 50.3 to 167.1 mg GAE/g DW among species. A positive linear correlation was observed between total phenolic content and antioxidant activity of the extracts. The results suggest that the extract of Salvia species, notably Salvia verticillata with the highest antioxidant activity, can be used as natural antioxidants in the food industry. <![CDATA[<b>Hormesis response of marine and freshwater luminescent bacteria to metal exposure</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200006&lng=en&nrm=iso&tlng=en The stimulatory effect of low concentrations of toxic chemicals on organismal metabolism, referred to as hormesis, has been found to be common in the widely used luminescence bioassay. This paper aims to study the hormesis phenomenon in both marine and freshwater luminescent bacteria, named Photobacterium phosphorem and Vibrio qinghaiensis. The effects of Cu (II), Zn (II), Cd (II) and Cr (VI) on luminescence of these two bacteria were studied for 0 to 75 minutes exposure by establishing dose- and time-response curves. A clear hormesis phenomenon was observed in all four testing metals at low concentrations under the condition of luminescence assays. <![CDATA[<b>Analysis of the intronic single nucleotide polymorphism rs#466452 of the nephrin gene in patients with diabetic nephropathy</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200007&lng=en&nrm=iso&tlng=en We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivaríate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site. <![CDATA[<b>Biological effects of an aqueous extract of <i>Salix alba </i>on the survival of <i>Escherichia coli </i>AB1157 cultures submitted to the action of stannous chloride</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200008&lng=en&nrm=iso&tlng=en Stannous chloride (SnC12) is used in nuclear medicine as a reducing agent to obtain technetium-99m-radiopharmaceuticals. It have been reported that natural products might reduce the genotoxic and cytotoxic effects related to SnC12. This work evaluated the biological effects of an aqueous extract of Salix alba on the survival of Escherichia coli (E. coli) AB1157 (wild type) cultures submitted to the action of SnC12. E. coli AB1157 cultures (exponential growth phase) were collected by centrifugation, washed and resuspended in 0.9%NaCl. Samples were incubated in water bath shaker with: (a) SnC12 (25mg/ml), (b)Salix alba extract(11.6mg/ml) and (c)SnC12(25mg/ml) + Salix alba extract (11.6mg/ml). Incubation with 0.9% NaCl was also carried out (control). At 60 min intervals, aliquots were withdrawn, diluted, spread onto Petri dishes with solid LB medium and incubated overnight. The colonies formed were counted and the survival fractions calculated. The extract was not able to protect the E. coli cultures against the lesive action of SnC12. The extract also did not interfere with the survival of the cultures. It suggested that the substances present in the Salix alba aqueous extract did not interfere strongly with cellular metabolism and did not alter the survival fractions of E. coli AB 1157. It is speculated that this extract cannot interfere with the generation of free radicals, the possible main agent responsible for SnC12 lesive action. <![CDATA[<b>Defense mechanisms of <i>Solanum tuberosum </i>L. in response to attack by plant-pathogenic bacteria</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200009&lng=en&nrm=iso&tlng=en The natural resistance of plants to disease is based not only on preformed mechanisms, but also on induced mechanisms. The defense mechanisms present in resistant plants may also be found in susceptible ones. This study attempted to analyze the metabolic alterations in plants of the potato Solanum tuberosum L. cv. Agata that were inoculated with the incompatible plant-pathogenic bacteria X. axonopodis and R. solanacearum, and the compatible bacterium E. carotovora. Levels of total phenolic compounds, including the flavonoid group, and the activities of polyphenol oxidase (PPO) and peroxidase (POX) were evaluated. Bacteria compatibility was evaluated by means of infiltration of tubers. The defense response was evaluated in the leaves of the potato plants. Leaves were inoculated depending on their number and location on the stem. Multiple-leaf inoculation was carried out on basal, intermediate, and apical leaves, and single inoculations on intermediate leaves. Leaves inoculated with X. axonopodis and with R. solanacearum showed hypersensitive responses within 24 hours post-inoculation, whereas leaves inoculated with E. carotovora showed disease symptoms. Therefore, the R. solanacearum isolate used in the experiments did not exhibit virulence to this potato cultivar. Regardless of the bacterial treatments, the basal leaves showed higher PPO and POX activities and lower levels of total phenolic compounds and flavonoids, compared to the apical leaves. However, basal and intermediate leaves inoculated with R. solanacearum and X. axonopodis showed increases in total phenolic compounds and flavonoid levels. In general, multiple-leaf inoculation showed the highest levels of total phenolics and flavonoids, whereas the single inoculations resulted in the highest increase in PPO activity. The POX activity showed no significant difference between single- and multiple-leaf inoculations. Plants inoculated with E. carotovora showed no significant increase in defense mechanisms such as enzyme activity and phenolic compounds. Therefore, resistance or susceptibility in S. tuberosum cv. Agata might depend on leaf age, type of inoculation performed (single or multiple), and the interaction between plant and pathogen. <![CDATA[<b>Analysis of the nuclear localization signal of TRF1 in non-small cell lung cancer</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200010&lng=en&nrm=iso&tlng=en Several studies revealed a similar down-regulation of telomeric repeat binding factor 1 (TRF1) in tumors. We have previously reported the TRFl expression levels were down-regulation in non-small cell lung cancer (NSCLC). The regulation of TRFl localization is proposed to be important for the function and expression. The nuclear localization signal (NLS) and nuclear export signal (NES) are often important clues to localization of protein. The objective of the present study was to investigate the NLS and NES of TRFl in NSCLC patients. Thirty (30) patients with NSCLCs had undergone radical operations in The First Affiliated Hospital, College of Medicine, Zhejiang University. DNA sequences of NLSs and NESs were amplified by PCR. The PCR products were analyzed by DNA sequencing. There were four NLSs of the TRFl protein, including two monopartite and two bipartite NLSs. The NLSs sequences were included in 337KKERRVGTPQSTKKKKESRR356. The exon 8 and exon 9 of TRFl DNA were covered the NLS sequences. The sequences of predicted NESs were 11WMLDFLCLSL86 and 174NLLKLQALAV183, respectively. The exon 1, exon 3 and exon 4 of TRFl were covered the NES sequences. In NSCLCs, there was no a mutation, deletion, or substitution in NLS and NES of TRFl. We conclude that the NLS and NES sequences in NSCLCs patients did not have mutations. Down-expression of TRFl does not indicate gene mutation of NLS and NES in NSCLCs. <![CDATA[<b>Some considerations about the theory of intelligent design</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200011&lng=en&nrm=iso&tlng=en The so-called theory of intelligent design (ID) has gained a growing reputation in the Anglo-Saxon culture, becoming a subject of public debate. The approaches that constitute the core of this proposal, however, have been poorly characterized and systematized. The three most significant authors of ID are certainly Michael Behe, William Dembski and Stephen Meyer. Beyond the differences that can be distinguished in the work of each of them, the central fact in their arguments is the complexity of living organisms, which according to these authors, escapes any kind of natural explanation. In effect, according to the authors of ID, the irreducible complexity that can be detected in the natural world would allow to infer design in a scientifically valid way, even though many of them prefer to remain silent regarding the identity and attributes of the designer. We think that under this proposal, remains a deep epistemological confusion, since its very structure combines methodologies that are beyond the scope of historical and natural evolutionary theories. We also reject the claim that ID is a legitimate scientific theory, because it does not exhibit the classical characteristics that a scientific kind of knowledge must have. <![CDATA[<b>Biological properties of the Chilean native moss <i>Sphagnum magellanicum</i></b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200012&lng=en&nrm=iso&tlng=en An ethanol extract prepared from the gametophyte Chilean native moss Sphagnum magellanicum was dried out, weighed and dissolved in distilled water. This extract was then assayed for its antibacterial activity against the G(-) bacteria Azotobacter vinelandii, Erwinia carotovora subsp. carotovora, Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Vibrio cholerae, and the G(+) bacteria Staphylococcus aureus subsp. aureus, and Streptococcus type b. The growth of the cultures of E. carotovora subsp. carotovora, and V. cholerae was inhibited at a concentration of 581mg/ml of extract, while the cultures of E. coli, S. typhi and Streptococcus type b were inhibited at a concentration of 1.16 mg/mL of extract. The concentration of phenolic compounds was 4.294 mg/mL; the presence of vanillic, chlorogenic, syringic, caffeic, gallic, 3-4 hydrozybenzoic, p-coumaric and salicylic acids was identified using RP- High Pressure Liquid Chromatography. <![CDATA[<b>Effects of substrate water potential in root growth of <i>Agave salmiana </i>Otto ex Salm-Dyck seedlings</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200013&lng=en&nrm=iso&tlng=en The objective of this study was to test the hypothesis that root of maguey (Agave salmiana Otto ex Salm-Dyck) seedlings reacts during the first 24 h to low substrate water potential (Yw), by anatomical modifications. Three-4 cm root length seedlings were planted in vermiculite for 24 h at Yw between -0.03 and -2.35 MPa. Root dimensions, proline content and anatomy were evaluated. Substrate ψw between -0.65 and -2.35 MPa did not significantly affect longitudinal root growth. However, proline content significantly increased from 1.6 to 2.1 emoles mg-1. Significant reductions of transverse root area (41 %), thickness of mucilage covering the epidermis (47 %), thickness of epidermis (between 15 and 46 %), area of the parenchyma (between 35 and 41 %) and number of vessels (up to 28 %) were observed with Yw of -2.35 MPa. In contrast, thickness of xylem wall, diameter of xylem vessels and the number of cells of the cortex of the differentiation root region significantly increased (64, 17, and 97 %, respectively). The anatomical changes associated with low substrate Yw indicate a net increase of root apoplatic paths; structures involved in water conduction increased their diameter under low substrate Yw conditions and anatomical changes occurred during the first 24 h of water stress. <![CDATA[<b>The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200014&lng=en&nrm=iso&tlng=en Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers. <![CDATA[<b>Fe de Erratas</b>]]> https://scielo.conicyt.cl/scielo.php?script=sci_arttext&pid=S0716-97602009000200015&lng=en&nrm=iso&tlng=en Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.