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Biomedical subjects

D Carbonera

Publications and source records attributed to D Carbonera.

At least 19 recordsLinked to original sources
PubMed2004

Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion.

The aim of the present study was to determine early cyto-histological events associated with the reduced number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers treated with methyl jasmonate (MJ) [S. Biondi et al. (2001) J Exp Bot 52:1-12]. The results show that 0.1-10 microM MJ strongly stimulated mitotic activity early in culture relative to untreated controls. Treatment with MJ also induced anomalous mitoses. Enhanced proliferative growth was confirmed by northern analysis and in situ hybridisation using cDNA probes of the G1/S phase-specific genes ubiquitin carboxyl-extension protein (ubi-CEP), topoisomerase 1 (top1) and ribonucleotide reductase (RNR). The formation of meristematic cell clusters on day 5 was also enhanced by 1 muM MJ, but subsequent development of these cell clusters into meristemoids and shoot primordia was reduced by all MJ concentrations in a dose-dependent manner. Cell expansion was stimulated by MJ concentrations ranging from 0.001 to 10 microM; expanded cells frequently occurred around and within meristemoids and shoot primordia, and displayed thickened and suberised cell walls; cell wall thickness increased with increasing MJ concentration. These cytological events caused alterations in the tunica and stem differentiation of the shoot dome. The apparently paradoxical role of MJ, which deregulates shoot formation through a stimulation of growth events, i.e., mitotic activity and cell expansion, is discussed.

Acetates↗
PubMed2001

Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I.

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47-RC in studying primary reactions of oxygenic photosynthesis.

Blotting, Western↗
PubMed2001

Spatial expression of DNA topoisomerase I genes during cell proliferation in Daucus carota.

The spatial expression of carrot (Daucus carota L.) top1 genes encoding the two isoforms of the enzyme DNA topoisomerase I (EC 5.99.1.2) was investigated. In situ hybridization analysis performed with a probe recognizing both top1 transcripts provided evidence that in explanted hypocotyls induced to proliferate in vitro by the addition of the growth regulator 2,4-dichlorophenoxyacetic acid (2,4-D), the mRNA accumulation parallels the proliferation of provascular cells of the stelar cylinder. During somatic embryogenesis, the histological distribution of top1 transcripts was strongly evident at the stage of torpedo-shaped embryos, but gene expression was not only restricted to meristematic regions. When the spatial localization was extended to carrot vegetative apices and the investigation was carried out with specific probes for top1alpha and top1beta, both transcripts preferentially accumulated in tissues having mitotic activity.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2000

Influence of the axial ligands on the spectral properties of P700 of photosystem I: a study of site-directed mutants.

Two histidines provide the axial ligands of the two chlorophyll a (Chl a) molecules which form the primary electron donor (P700) of photosystem I (PSI). Histidine 676 in the protein subunit PsaA, His(A676), and histidine 656 in subunit PsaB, His(B656), were replaced in the green algae Chlamydomnas reinhardtii by site-directed mutagenesis with nonpolar, uncharged polar, acidic, and basic amino acid residues. Only the substitutions with uncharged polar residues led to a significant accumulation of PSI in the thylakoid membranes. These PSI complexes were isolated and the physical properties of the primary donor characterized. The midpoint potential of P700(+)(*)/P700 was increased in all mutants (up to 140 mV) and showed a dependence on size and polarizability of the residues when His(B656) was substituted. In the light-minus-dark absorbance spectra, all mutations in PsaB exhibited an additional bleaching band at 665 nm at room temperature comparable with the published spectrum for the replacement of His(B656) with asparagine [Webber, A. N., Su Hui, Bingham, S. E., Käss, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E., and Lubitz, W. (1996) Biochemistry 35, 12857-12863]. Substitutions of His(A676) showed an additional shoulder around 680 nm. In the low-temperature absorbance difference spectra of P700(+)(*)/P700, a blue shift of the main bleaching band by 2 nm and some changes in the spectral features around 660 nm were observed for mutations of His(B656) in PsaB. The analogous substitution in PsaA showed only a shift of the main bleaching band. Similar effects of the mutations were found in the (3)P700/P700 absorbance difference spectra at low temperatures (T = 2 K). The zero-field splitting parameters of (3)P700 were not significantly changed in the mutated PSI complexes. The electron spin density distribution of P700(+)(*), determined by ENDOR spectroscopy, was only changed when His(B656) was replaced. In all measurements, two general observations were made. (i) The replacement of His(B656) had a much stronger impact on the physical properties of P700 than the mutation of His(A676). (ii) The exchange of His(B656) with glutamine induces the smallest changes in the spectra or the midpoint potential, whereas the other replacements exhibited a stronger but very similar influence on the spectroscopic features of P700. The data provide convincing evidence that the unpaired electron in the cation radical and the triplet state of P700 are mainly localized on the Chl a of the dimer which is axially coordinated by His(B656).

Amino Acid Substitution↗
PubMed2000

Carrot cells contain two top1 genes having the coding capacity for two distinct DNA topoisomerases I.

Five DNA topoisomerase I cDNA clones were isolated from a carrot (Daucus carota) cDNA library and two classes of nucleotide sequences were found. One component of the first class, pTop9, perfectly matches the open reading frame of pTop28, a truncated top1 cDNA previously described, and extended it by 594 nucleotides (top1alpha). A member of the second class, pTop11, contains an open reading frame 2727 bp long (top1ss) with a coding capacity for a second putative DNA topoisomerase I of 101 kDa. Both pTop9 and pTop11 clones are full length cDNAs. The two deduced amino acid sequences share a relevant similarity (89%) only at the C-terminal domain, whereas the similarity is reduced to 32% in the N-terminal region. Southern blot analysis and PCR amplification of genomic DNAs from carrot pure lines suggested the presence of two distinct loci. Northern blot analysis revealed the presence of two distinct transcripts of 3.0 and 3.2 kb in both cycling and starved cell populations. Three fusion peptides corresponding to the N-terminal domain of the alpha and ss forms and from the common C-terminal domain of carrot topoisomerases I were overexpressed in E. coli cells and used to raise antibodies in rabbit. Immunolocalization seems to suggest the presence of two topoisomerases I in carrot nuclei.

Amino Acid Sequence↗
PubMed1996

Cloning of a cDNA encoding DNA topoisomerase I in Daucus carota and expression analysis in relation to cell proliferation.

DNA topoisomerase I is an enzyme involved in several processes related to DNA metabolism. Despite the physiological importance, the regulation of top1 gene expression has not yet been investigated in plants. In order to monitor the possible correlation between levels of top1 transcripts and the proliferative state of the cell, two partially overlapping cDNAs encoding DNA topoisomerase I from Daucus carota have been isolated from a poly(A)(+)-primed library, using an Arabidopsis thaliana probe, and from a cDNA library spanning the 5' region of the top1 transcript, which was constructed using an antisense specific oligonucleotide. The top1 nucleotide sequence encoded an open reading frame of 2370 bp, predicting a protein of 90 kDa. The deduced amino acid sequence showed a similarity of 51% with A. thaliana, 41% with S. cerevisiae, 40% with S. pombe and 31% with H. sapiens, respectively. Southern blot analysis, performed under moderate stringency conditions, showed the presence of a single-copy gene. Evaluation of the top1 mRNA steady-state level revealed, besides a constitutive expression in vegetative carrot tissues, an induced expression related to cell proliferation.

Amino Acid Sequence↗
PubMed1994

A well resolved ODMR triplet minus singlet spectrum of P680 from PSII particles.

An ADMR T-S spectrum of the primary donor (P680) of photosystem II (PSII) was obtained from anaerobically photoreduced particles. The spectrum is the best resolved obtained so far having a main bleaching band at 684 nm with a linewidth of only 100 cm-1. The view that this spectrum is produced by native homogeneous P680 unlike those obtained before is defended. A small bleaching observed at 678 nm is discussed in terms of the reaction center structure. One possible interpretation of the observations is that P680 is a very loose dimer with an exciton splitting of only 144 cm-1 corresponding to a dimer center-to-center distance of roughly 11.5 A.

Chlorophyll↗
PubMed1991

Proteolytic and partial sequencing studies of the bifunctional dihydrofolate reductase-thymidylate synthase from Daucus carota.

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) of Daucus carota has been further characterized as regards molecular weight, amino acid composition, protease digestion and microsequencing of proteolytic peptides. Data reported in this paper demonstrate that the carrot protein has a calculated Mr of 124,000 thus indicating that, contrarily to what has previously been suggested, it occurs as a dimer of identical subunits. Results of partial amino acid microsequencing show the presence of sequences highly homologous with those of the active sites of both DHFR and TS from other organisms confirming, at the structural level, the bifunctional nature of the carrot protein. As in the case of Leishmania tropica DHFR-TS, incubation of the carrot protein with V8 protease led to a rapid loss of TS activity while retaining that of DHFR. However the pattern of proteolysis did not allow to establish whether the sequence of domains is DHFR-TS as in Leishmania, or vice versa. Low homology of other amino acid sequences, as judged by computer analysis, and absence of common epitopes indicate an apparent divergence between carrot and leishmanian proteins.

Amino Acid Sequence↗
PubMed1991

Cellular localization of dihydrofolate reductase-thymidylate synthase in carrot cells.

The intracellular distribution and localization of dihydrofolate reductase-thymidylate synthase of wild type suspension carrot cells was analysed using cytochemical and immunocytochemical techniques; in both resting and growing normal cells (E4) the activity appeared to be predominantly cytoplasmic. The pattern of localization of the enzyme was also analysed during the different phases of the cell cycle. To this end carrot cells were synchronized with aphidicolin (an inhibitor of the alpha-like DNA polymerase which blocks cells at the G1/S boundary) and cycle phases checked by labelled-thymidine incorporation. Protoplasts obtained from cells inhibited with aphidicolin or from cells sampled at different times after the removal of the drug (S and G2 phase), failed to show a nuclear localization of DHFR-TS. These results indicate that in carrot the bifunctional enzyme does not change compartment during the cell cycle. Surprisingly Mtx-resistant cells (E2A2, E2A1C6; overproducing DHFR-TS) showed, irrespective of their physiological state (quiescent or growing), also a relevant nuclear or perinuclear immunofluorescence which could not be detected using cytochemical techniques. The reason of this altered localization is not clear and its possible relation with altered cytophysiological parameters is discussed.

Cell Cycle↗
PubMed1988

Mechanism of nitrofurantoin toxicity and oxidative stress in mitochondria.

5-Nitrofuran derivatives change the inner mitochondrial membrane permeability as indicated by the transmembrane potential, the rate of spontaneous K+ efflux and the basal respiratory rate: (a) at low concentrations nitrofurantoin prevents the increase of inner membrane permeability due to hydroperoxides or to diamide; (b) at higher concentrations or after longer times of incubation, nitrofurantoin enhances the membrane damage due to hydroperoxides or to diamide; the damage due Ca2+ plus Pi is enhanced by nitrofurantoin at all concentrations; (c) higher nitrofurantoin concentrations cause membrane damage independently of the presence of hydroperoxides or of diamide. The effect of nitrofurantoin is cancelled by the addition of free-radical scavengers. The above effects of nitrofurantoin are compatible with the observations of Mason and colleagues that nitrofurantoin is reduced by a NADPH nitroreductase to a nitro anion radical which can then undergo subsequent reactions, among which are (a) initiation of a free-radical reaction chain and (b) reduction of hydroperoxides and diamide.

Animals↗
PubMed1988

Permeability of inner mitochondrial membrane and oxidative stress.

The mechanism of increase in the inner membrane permeability induced by Ca2+ plus Pi, diamide and hydroperoxides has been analyzed. (1) The permeability increase is antagonized by oligomycin and favoured by atractyloside. The promoting effect of atractyloside is strongly reduced if the mitochondria are simultaneously treated with oligomycin. (2) Addition of the free-radical scavenger, butylhydroxytoluene, results in a complete protection of the membrane with respect to the permeability increase. (3) Although membrane damage and depression of the GSH concentration are often associated, there is no direct correlation between extent of membrane damage and concentration of reduced glutathione. Abolition of the permeability increase by butylhydroxytoluene or by oligomycin is not accompanied by maintenance of a high GSH concentration in the presence of diamide or hydroperoxides. The membrane damage induced by Ca2+ plus Pi is not accompanied by a depression of the GSH concentration. (4) It is proposed that a variety of processes causing an increased permeability of the inner mitochondrial membrane merge into some ultimate common steps involving the action of oxygen radicals.

Adenosine Triphosphate↗
PubMed1987

Production and Preliminary Characterization of Monoclonal Antibodies against Cationic Peanut Peroxidase.

Ten monoclonal antibodies (McAbs) have been produced against the cationic peroxidase from peanut suspension cell culture. Eight of these antibodies were found to be of the immunoglobulin (Ig)G(1) subclass and two were of IgA subclass. A combination of competitive enzyme-linked immunosorbent assay, Western blotting analysis, and direct antigen-binding assay revealed that the antibodies are directed against four different epitopes on the cationic peroxidase and the McAbs can be subdivided into four groups. Only group A inhibits peroxidase activity. Group B and D bind equally well to the native and the denatured form of cationic peroxidase, whereas the remaining McAbs react with more or less reduced affinity to the denatured antigen. Group C probably recognizes a conformation-dependent epitope. All the McAbs cross react weakly with the anionic peanut peroxidase, suggesting a structural nonidentity as well as some similarity between these two peroxidase isozymes. Cross reactivities of these McAbs with peroxidases of various plant species were also demonstrated.

Journal Article↗
PubMed1979

[Myasthenia gravis. Indications, aims and methods of intensive therapy].

Some recent views on pathophisyology of "myasthenia gravis" are presented. The Authors explain the typical damage of myasthenia gravis, i.e. the progressively reduced muscolar function on the basis of an autoimmune derangement affecting the motor-end plates. The most commonly used types of treatment, both medical (i.e. antiChE, A.C.T.H., steroids and immunodepressant drugs) and surgical (thymectomy) are reviewed. The very important role of Intensive Care for the treatment either of myasthenia gravis "per se" or of possible consequences of some drugs (A.C.T.H. steroids) is also stressed. Finally the Authors present their results about their experience on 36 patients affected by myasthenia gravis and admitted to I.C.U. once (29 patients) or twice or more (7 patients). The Authors describe some practical problems presented by patients during their stay in I.C.U.

Adolescent↗