Vol.29-Issue 2 - 2005
Vol.29-Issue 2 - 2005
Review
Mitochondrial apoptotic pathways
NORA MOHAMAD, ALICIA GUTIÉRREZ, MARIEL NÚÑEZ, CLAUDIA COCCA, GABRIELA MARTÍN*, GRACIELA CRICCO,
VANINA MEDINA, ELENA RIVERA, AND ROSA BERGOC*.
Radioisotopes Laboratory, School of Pharmacy and Biochemistry, University of Buenos Aires. Buenos Aires, Argentina.
* Members of CONICET
Key words: apoptosis, mitochondria, Bcl-2 family, cytochrome-c.
ABSTRACT: Apoptosis or programmed cell death (PCD) is a physiological process characteristic of pluricellular organisms leading to self-destruction of the cell. It is therefore involved in development, homeostasis and host defense. However, a significant difference has been shown between mammalian cell apoptosis and non-mammalian cell apoptosis: mitochondria are implicated only in the former. Execution of PCD includes the release of several proapoptotic proteins from the intermembrane space of mitochondria. They could exert their actions through a caspase dependent as well as a caspase independent way. On the other hand, regulation of PCD is mainly given by the Bcl-2 family members, which are in turn essentially regulated by activation of death receptors and/or DNA damage. Nowadays, execution of apoptosis is better known than its regulation. Nevertheless, we are still far of a complete understanding of the apoptotic process.
Abbreviations: ADP, Adenosine diphosphate; AIF, Apoptosis-Inducing Factor; Akt, homologues of the v-akt oncogene;
ANT, Adenine Nucleotide Translocator; Apaf-1, Apoptosis protease-activating factor-1; ATP, Adenosine triphosphate;
Bad, Bak, Bax, Bid, Bim, Members of the Bcl-2 family; Bcl-2, B cell lymphoma-2 protein; BH, Bcl-2 homologue; DIABLO,
Direct IAP binding Protein with low pI; DNA, deoxiribonucleic acid; FADD, Fas-associated death domain protein; Fas,
receptor of death that belongs to the TNF receptor (TNFR) family; Fas-L, Fas ligand; HSPs, Heat Shock Proteins; HtrA,
High-temperature requirement; IAP, Inhibitor of Apoptosis Protein; IMM, Inner Mitochondrial Membrane; IMS, Intermembrane
Space; MA, Mitochondrial Anchorage; Noxa, p53-inducible (BH3)-only protein; Omi/HtrA2, mammalian serine
protease; OMM, Outer Mitochondrial Membrane; PCD, Programmed Cell Death; PTP, Permeability Transition Pore; Puma,
p53- inducible (BH3)-only protein; ROS, Reactive Oxigen Species; SMAC, Second Mitochondrial Activator of Caspase;
TNF-a, Tumor Necrosis Factor alpha; TNF-R, Tumor Necrosis Factor Receptor; TRADD, TNF-receptor-1 associated death
domain protein; VDAC, Voltage-Dependent Anion Channel.
Structure and secretory activity of cultured chondrocytes from
patients with osteoarthritis
HILDA L. MONTRULL, NILDA Y. BRIZUELA, SILVIA L. DEMURTAS, LUIS SPITALE* AND CARLOS I. MEIROVICH.
Departamento de Farmacología, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Argentina.
* II Cátedra de Anatomía y Fisiología Patológica, Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Argentina.
Keywords: Osteoarthritis, chondrocytes, collagenase.
ABSTRACT: Cartilage samples were taken from OA patients in order to describe and quantify pro-inflammatory
mediators. Samples were cultured under aseptic conditions in Dulbecco’s modified Eagle medium at
37°C for 10 days. Control samples, taken from non-inflammatory cartilage, were cultured under the same
conditions. The levels of NO-
2 and NO-
3 were measured in the supernatant using a spectrophotometric assay.
The activity of MMP-1 was quantified by ELISA.
The concentration of NOx
was 47.3 ± 4.1 µM in the OA cartilague and 10.7 ± 1.8 µM in the controls.
The average MMP-1 activity was 3,650 ± 387 ng/ml in the OA cartilage and 2,150 ± 190 ng/ml in the control
samples. These increased values of MMP-1 and NOx
observed in the OA cartilage suggest a higher catabolic
activity.
A morphological analysis of OA chondral tissue using light microscopy shows that the surface of the
tissue is characterized by the presence of aggregated chondrocytes or “clones” but in the deeper areas isolated
cells are found.
These results could be a significant contribution towards the identification of biological markers indicating
the presence of OA activity.
DNA injury induced by 5-aminouracil and caffeine in G2
checkpoints path of higher plant cells
A. DEL CAMPO*, M. BRACHO, L. MARCANO, J. GUÍÑEZ, AND C. DE LA TORRE**.
* Universidad del Zulia. Facultad Experimental de Ciencias. Departamento de Biología. Maracaibo. Estado Zulia. Venezuela.
** Consejo Superior de Investigaciones Científicas. Centro de Investigaciones Biológicas. Departamento de Biología Celular
y del Desarrollo. Madrid, España.
Key words: 5-aminouracil, G2 checkpoints, caffeine.
ABSTRACT: This work evaluated the qualitative and quantitative cellular changes induced by treatment
with 5-aminouracil (5-AU) and a combination of 5-AU and caffeine in plant cells in relation to DNA damage,
repaired damage, and residual damage. As biological material, Allium cepa L. root tips were used, grown in
filtered water, in darkness, with aeration at constant temperature of 25 °C ± 0.5. Cell populations were
synchronized using 5 mM caffeine in order to study the effects of 5-AU and caffeine/5-AU combined treatment
on the DNA content and their incidence in the entrance to mitosis. The results showed a delay in the G2
period due to induced DNA damage by the 5-AU and caffeine/5-AU combined treatment, shown by aberrant
metaphases, anaphases and telophases. The effect of caffeine in the combined treatment was heightened in
spite of lengthening the checkpoints route that retains the cells in G2. The existence of G2 checkpoints was
shown in the cell population studied, inducing lesions in the DNA, chromosomic aberrations and cellular
instability.
A male-sterile mutation in soybean (Glycine max) affecting
chromosome arrangement in metaphase plate and cytokinesis
NILTON CESAR PIRES BIONE1, MARIA SUELY PAGLIARINI2 AND LEONES ALVES DE ALMEIDA3
1 Department of Sciences, Unicentro, 84500-000, Irati PR Brazil.
2 Department of Cell Biology and Genetics, State University of Maringá, 87020-900 Maringá PR Brazil.
3 Embrapa-National Soybean Research Center (Embrapa Soybean), Londrina PR Brazil.
Key words: Glycine max, meiosis, metaphase plate, cytokinesis, male sterility
ABSTRACT: A spontaneous male-sterile, female-fertile mutation affecting bivalent arrangement at the metaphase plate and cytokinesis was detected in line BR98-197 of the soybean breeding program developed by Embrapa – National Soybean Research Centre. Untill diakinesis, meiosis was normal with chromosome pairing as bivalents. From this phase, in several meiocytes, bivalents were not able to organize a single metaphase plate and remained scattered in the cytoplasm in a few or several groups. In these meiocytes, chromosomes segregated in both divisions giving rise to several micronuclei. However, the main cause of male sterility was the absence of cytokinesis after telophase II. Instead of the typical tetrads of microspores, four nucleate coenocytic microspores were formed. In the mutant, pollen mitoses did not occur, and after engorgement by starch, pollen underwent a progressive process of degeneration.
Regulation of mouse embryo development by autocrine
throphic factors
M.T. TERUEL1-2, R.C. CATALANO2, J.A. CABODEVILA2 AND S.S. CALLEJAS2
1Area de Embriología. 2Area de Reproducción, FISFARVET. Facultad de Ciencias Veterinarias, Universidad Nacional del
Centro de la Provincia de Buenos Aires, (B7000GHG) Tandil, Argentina.
Key words: embryo development, mouse, autoregulation, trophic factors.
ABSTRACT: Embryo development depends on maternal and embryonic factors. When occurs in vitro,
embryos secrete factors that stimulate their development. The purpose of this study was to investigate the
possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned
media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-F10 culture
medium during 24 h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and
CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between
embryos cultured for 24h in Ham-F10, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However,
CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-F10 medium
(80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to
Ham-F10 evaluated at 78 and 96 h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After
72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that
in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture,
differences between CM5, CM10 and Ham-F10 were not observed. These results suggest that preimplantational
mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process.
Molecular cloning and characterization of a novel mannosebinding
lectin cDNA from Zantedeschia aethiopica
ZHONGHAI CHEN1, YONGZHEN PANG1, XIAOJUN LIU1, XINGLONG WANG1, ZHONGXIANG DENG1, XIAOFEN SUN1
AND KEXUAN TANG1,2
1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Morgan-Tan International Center for Life Sciences,
Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Fudan University, Shanghai 200433, People’s Republic
of China.
2. Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture
and Biology, Shanghai Jiaotong University, Shanghai 200030, People’s Republic of China.
Key words: Mannose-binding lectin, RACE, Zantedeschia aethiopica, ZAA
ABSTRACT: Using RNA extracted from Zantedeschia aethiopica young leaves and primers designed according to the conservative regions of Araceae lectins, the full-length cDNA of Z. aethiopica agglutinin (ZAA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zaa was 871 bp and contained a 417 bp open reading frame (ORF) encoding a lectin precursor of 138 amino acids. Through comparative analysis of zaa gene and its deduced amino acid sequence with those of other Araceae species, it was found that zaa encoded a precursor lectin with signal peptide. Secondary and three-dimensional structure analyses showed that ZAA had many common characters of mannose-binding lectin superfamily and ZAA was a mannose-binding lectin with three mannose-binding sites. Southern blot analysis of the genomic DNA revealed that zaa belonged to a multi-copy gene family.
Changes in crossover distribution along a quadrivalent in a man
carrier of a reciprocal translocation t(11;14)
M.I. PIGOZZI, R.B. SCIURANO, AND A.J. SOLARI
Centro de Investigaciones en Reproducción (CIR), Facultad de Medicina, Paraguay 2155, (1121) Buenos Aires, Argentina.
Key words: Human meiotic recombination, human meiosis, crossovers, MLH1 foci, chromosomal translocations.
ABSTRACT: A testicular biopsy from an infertile man carrying a heterozygous chromosome translocation
t(11;14) was studied with synaptonemal complex analysis and immunolocalization of the protein MLH1 for
crossover detection. A full blockage of spermatogenesis at the spermatocyte stage was related to the presence
of the translocation quadrivalents at pachytene. Only 2% of the quadrivalents showed full synapsis. Most of
the spermatocytes showed asynaptic free ends that frequently mingled with the XY pair. The average number
of crossovers per cell was diminished from a mean of 52.7 in controls to a mean of 48 in the patient. The
difference between the number of crossovers in the quadrivalent and the normal bivalents was highly significant.
The distribution of crossovers over the segment of the quadrivalent corresponding to bivalent #14 was
also very different from that of the control. It is concluded that in this translocation, the pattern of crossovers
is changed, mainly due to a synaptic hindrance in the quadrivalent, and that the spermatogenesis arrest is
mainly due to the quadrivalents that interact with the XY pair.
Brief Note
Natural infection of Viola cornuta (Violaceae) with Cucumber
mosaic virus, subgroup I
JOEL ARNEODO*, SOLEDAD DE BREUIL*, SERGIO LENARDON** AND LUIS CONCI**
* Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
** Instituto de Fitopatología y Fisiología Vegetal (IFFIVE) – Instituto Nacional de Tecnología Agropecuaria (INTA)
Keywords: Viola cornuta, Cucumber mosaic virus, electron microscopy, serology, AC-RT-PCR.
ABSTRACT: Plants of Viola cornuta displaying typical virus symptoms were observed during spring 2003 in a plant nursery in Córdoba, central Argentina. Electron microscopic examinations of symptomatic leaf samples revealed the presence of isometric virus-like particles about 30 nm in diameter. Subsequent serological analysis allowed the identification of the pathogen as a subgroup I strain of Cucumber mosaic virus (CMV). These results were confirmed by antigen capture - reverse transcription - polymerase chain reaction with specific CMV primers, and digestion with a restriction enzyme. This is the first report of CMV infecting V. cornuta in Argentina.
Reactive oxygen species in bovine embryo in vitro production
G.C. DALVIT, P.D. CETICA, L.N. PINTOS AND M.T. BECONI
Area of Biochemistry, School of Veterinary Sciences, University of Buenos Aires.
Chorroarín 280, (C1427CWO) Buenos Aires, Argentina.
Key words: reactive oxygen species, oxidative stress, oocyte, embryo, bovine
ABSTRACT: Oxidative modifications of cell components due to the action of reactive oxygen species (ROS) is one of the most potentially damaging processes for proper cell function. However, in the last few years it has been observed that ROS participate in physiological processes. The aim of this work was to determine ROS generation during in vitro production of bovine embryos. Cumulus-oocyte complexes were recovered by aspiration of antral follicles from ovaries obtained from slaughtered cows and cultured in medium 199 for 22 h at 39ºC in 5% CO2: 95% humidified air. In vitro fertilization was carried out in IVF-mSOF with frozenthawed semen in the same culture conditions and embryo in vitro culture in IVC-mSOF at 90% N2: 5% CO2: 5% O2. ROS was determined in denuded oocytes and embryos at successive stages of development by the 2´,7´-dichlorodihydrofluorescein diacetate fluorescent assay. ROS production was not modified during oocyte maturation. However, a gradual increase in ROS production was observed up to the late morula stage during embryo in vitro culture (P<0.05). In expanded blastocysts, ROS level decreased to reach values similar to the corresponding in oocytes. In the bovine species, the variation in ROS level during the complete process of embryo in vitro production was determined for the first time.