Biomedical subjects

R Marco

Publications and source records attributed to R Marco.

At least 19 recordsLinked to original sources

PubMed1998

Differential muscle-type expression of the Drosophila troponin T gene. A 3-base pair microexon is involved in visceral and adult hypodermic muscle specification.

The complete genomic organization of the Drosophila troponin T (TnT) gene shows many interesting features, including the presence of a microexon of only 3 nucleotides conserved among Drosophilidae. It is the smallest bona fide exon so far described, placing a new lower limit on the nucleotide number required for correct splicing. Four muscle-type specific transcripts are generated by developmentally regulated alternative splicing. Exons 3, 4, and 5 are absent in the transcript present in jump and flight muscles. A total of 11 exons are present in the adult hypodermic muscles transcript, whereas the microexon is absent in the larval hypodermic musculature. The two isoforms differ in a lysine residue. Post-translational regulation of the flight muscles/tergal depressor of the trochanter-specific isoform is involved in flight and/or jump function. The interaction domains of TnT in the tropomyosin-troponin complex are strongly conserved in the known vertebrate and invertebrate TnT sequences, whereas the terminal regions show an important variability. The COOH-terminal region shows important phylogenetic variations, whereas the NH2-terminal domain is associated with specific muscle types in a particular organism, a finding that discloses a selective value for these domains in the functionality of distinct muscles in different organisms.

Amino Acid Sequence↗

PubMed1998

Troponin-T is a calcium-binding protein in insect muscle: in vivo phosphorylation, muscle-specific isoforms and developmental profile in Drosophila melanogaster.

Two sets of muscle polypeptides showing calcium-binding capacity and intense labelling in vivo with 32P were purified and characterized from Drosophila melanogaster adult extracts. The polypeptides exhibit crossed immunoreactivity and share similar biochemical properties such as those involved in purification. They have been identified as isoforms of troponin-T (TnT) by sequence analysis of a cDNA clone isolated from an embryonic library. The two sets of TnT polypeptides correspond to the fibrillar and non-fibrillar muscle isoforms, respectively. The non-fibrillar muscle isoforms separate into two bands which are differentially expressed during development. Analysis of TnT isoforms in bee thoraces indicates that the expression of the fibrillar muscle isoform correlates with the acquisition of functional flight capability. In vivo labelling experiments reveal that the two TnT sets are readily phosphorylated. The Drosophila TnTs show calcium-binding properties by three different types of assays. Our results suggest that this property could be specific to insect TnTs and may be related to the long, extremely acidic polyglutamic carboxy-terminus present in these polypeptides, which does not occur in non-arthropod TnTs.

Amino Acids↗

PubMed1997

Artemia mitochondrial genome: molecular biology and evolutive considerations.

During the last two decades an increasing amount of information has been accumulated regarding the gene structure and organization of the mitochondrial genome from various organisms. Many studies carried out mainly in mammals, have contributed to the knowledge of the basic elements involved in the replication and transcription of mitochondrial DNA. However, very little is known about these processes in invertebrates. In this review we discuss our current knowledge of the animal mitochondrial genetic system and briefly summarize the structure of the Artemia mitochondrial genome, the characteristics of its transcriptional machinery and how its expression is controlled during early development, in relation with what is known in other organisms. Artemia is the only crustacean where the mtDNA has been studied at this level of detail up to date.

Animals↗

PubMed1997

Biodynamics of attachments used in overdentures: experimental analysis with photoelasticity.

Treatment with overdentures is an alternative to conventional complete prostheses. Different types of attachments can be placed in natural teeth that act as retainers for overdentures. The photoelastic relationship between the design of the attachment and the distribution of occlusal forces among the abutment teeth and the distal alveolar ridges was examined. The results were used to develop a classification for clinical cases so that the correct attachment design can be selected.

Dental Abutments↗

PubMed1996

Preservation of viable biological samples for experiments in space laboratories.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

Animals↗

PubMed1996

Microgravity effects on Drosophila melanogaster behavior and aging. Implications of the IML-2 experiment.

Earlier Space experiments had indicated that young male Drosophila flies exposed to microgravity showed an acceleration in aging. In a 14.5-day Space Shuttle Flight we sent 300 young male flies with the purpose of confirming these findings and to establish whether changes in the behavior of the flies were responsible for the effect in accordance with the proposal that alterations in mitochondrial metabolism may be involved in the aging response. By repeatedly video-recording, we have found a very marked increase in the locomotor activity of the fruitflies in Space. The males showed an accelerated aging response upon recovery, both in terms of physiological vitality assays (mating and negative geotaxis) and of life-span curves. The involvement of mitochondrial metabolism is also suggested by the finding of a greater decrease in mitochondrial 16S ribosomal RNA in the microgravity exposed flies than in ground controls. On the other hand, a parallel 1 x g centrifuge control did not show such differences in the life-span curves when compared to flies exposed to a similar centrifugation on the ground. Drosophila females also increased their locomotor activity but did not show differential changes in the life-span curves. These results are discussed in terms of the current mechanisms of aging in multicellular eukaryotic organisms.

Aging↗

PubMed1996

Effects of the space environment on Drosophila melanogaster development. Implications of the IML-2 experiment.

One hundred and sixty Drosophila females laid several thousands of embryos during the 14.5 days of the IML-2 spaceflight. The progeny were either recovered frozen (embryos at final stages of development and larvae), or maintained alive developing further until adulthood. All embryos, larvae, pupae and imagoes recovered were normal in morphology and function. Results from earlier experiments were reproduced in IML-2 with a better experimental design. We confirm that in Space there is a stimulation of oogenesis and that development is slightly delayed when compared to that of synchronous parallel ground controls. Nevertheless, it is clear from the accompanying 1 x g flight control centrifuge and from the 1.4 x g ground centrifuge samples, that centrifugation itself can produce similar effects, emphasizing the importance of reevaluating the role of the 1 x g on-flight controls. The results emphasize the apparent paradox that simple cellular model systems in microgravity show alterations in many fundamental processes such as those involved in cell signalling, while development, relying heavily on these key cellular mechanisms can proceed quite normally in the absence of gravity. The effects on development are small and more the consequence of a reaction to the abnormal Space environment in general than a specific effect of microgravity.

Animals↗

PubMed1996

Study of the action of intramuscularly administered erythromycin on the L-threonine transport and the digestive enzymatic activity in rabbit jejunum.

Erythromycin has been shown to inhibit the intestinal transport of L-threonine and D-galactose in strips of mucosal jejunum when it was directly added to the incubation medium. Nevertheless, the effect of erythromycin administered therapeutically by intramuscular injection on both the intestinal absorption of nutrients and the intestinal digestive activity, remains unknown. The results obtained show that, firstly, the intestinal absorption of L-threonine is inhibited in animals treated with erythromycin. The kinetic study shows that the effect seems to be mainly due to an alteration of the affinity apparent constant (Kt) of the Na(+)-dependent system of transport located in the mucosal border. However, the Na(+)-dependent L-threonine transport in BBMV was not altered by the treatment with erythromycin. The (Na(+)-K+) ATPase activity in BLMV from treated jejunum was 40% of the activity in control BLMV. Secondly, the treatment with erythromycin did not modify the digestive enzymatic activity of sucrase and aminopeptidase N.

Aminopeptidases↗

PubMed1996

Serotonin-induced changes in L-leucine transport across rabbit jejunum.

The aim of the present study has been to determine the effect of serotonin (5-HT) on the absorption of L-leucine across the rabbit jejunum. The results show that serotonin significantly diminishes the uptake and steady-state tissue accumulation and the mucosal to serosal flux of L-leucine. This effect does not change with previous intestinal exposure of the mucosa to the 5-HT. Serotonin does not seem to modify amino acid simple diffusion across the intestinal epithelium. The effect on the amino acid uptake is due to an inhibition of the Na(+)-dependent system of transport, mainly through a reduction of the apparent Vmax. Moreover, this hormone, added to the incubation solution, does not affect the L-leucine uptake across brush border membrane vesicles. In presence of trifluoroperazine, TMB-8 and staurosporine, the serotonin effect disappears. These results suggest an effect mediated by intracellular processes related to protein kinase C which inhibit the intestinal absorption of L-leucine.

Animals↗

PubMed1996

Study of the binding of motilin to the membranes of enterocytes from rabbit jejunum.

The results obtained in the present work have shown that [125I]motilin bound specifically to basolateral (BL) membrane but it did not bind to the brush border (BB) membrane of the rabbit jejunum enterocyte. The [125I]motilin dissociation constant (Kd) was 95.58 +/- 15.0 pM and the receptor density (Bmax) was 2.54 +/- 0.40 fmol/mg protein. The binding of [125I]motilin to BL membrane was competitively inhibited by both unlabeled motilin and erythromycin. The IC50s were (2.1 +/- 0.4) 10(-8) M and (1.3 +/- 0.1) 10(-6) M for motilin and erythromycin, respectively, and the Ki were (6.83 +/- 1.3) 10(-9) M for motilin and (4.32 +/- 0.33) 10(-7) M for erythromycin. Saturation and competition binding studies showed interaction at only one class of binding sites in BL membrane.

Animals↗

PubMed1996

Drosophila paramyosin/miniparamyosin gene products show a large diversity in quantity, localization, and isoform pattern: a possible role in muscle maturation and function.

The Drosophila paramyosin/miniparamyosin gene expresses two products of different molecular weight transcriptionally regulated from two different promoters. Distinct muscle types also have different relative amounts of myosin, paramyosin, and miniparamyosin, reflecting differences in the organization of their thick filaments. Immunofluorescence and EM data indicate that miniparamyosin is mainly located in the M line and at both ends of the thick filaments in Drosophila indirect flight muscles, while paramyosin is present all along the thick filaments. In the tergal depressor of the trochanter muscle, both proteins are distributed all along the A band. In contrast, in the waterbug, Lethocerus, both paramyosin and miniparamyosin are distributed along the length of the indirect flight and leg muscle thick filaments. Two-dimensional and one-dimensional Western blot analyses have revealed that miniparamyosin has several isoforms, focusing over a very wide pH range, all of which are phosphorylated in vivo. The changes in isoform patterns of miniparamyosin and paramyosin indicate a direct or indirect involvement of these proteins in muscle function and flight. This wide spectrum of potential regulatory characteristics underlines the key importance of paramyosin/miniparamyosin and its complex isoform pattern in the organization of the invertebrate thick filament.

Animals↗

PubMed1995

Analysis of the paramyosin/miniparamyosin gene. Miniparamyosin is an independently transcribed, distinct paramyosin isoform, widely distributed in invertebrates.

Miniparamyosin, a distinct Drosophila melanogaster paramyosin isoform of 60 kDa, is shown here to be encoded by the same gene as paramyosin. The gene, located at 66D14, spans over 12.8 kilobases (kb) and is organized into 10 exons, 9 of which code for the paramyosin transcripts. An exon, located between exons 7 and 8, codes for the 5'-end of the miniparamyosin, and the two proteins share the two last exons of the gene. Mapping of the 5'-ends of these transcripts indicates that the paramyosin and miniparamyosin mRNAs arise from two overlapping transcriptional units; the miniparamyosin transcription initiation site is located inside a paramyosin intron, 8 kb downstream of the one used for paramyosin transcription. The existence of two different promoters and the conserved and nonconserved features of their sequences suggest a very complex regulation of these two muscle proteins. In fact, while paramyosin is expressed at two distinct stages of development as most other Drosophila muscle proteins, miniparamyosin appears late in development, being present only in the adult musculature. The absence of exon 1B, the specific exon of miniparamyosin, in the nematode Caenorhabditis elegans, as well as additional lines of evidence support the lack of miniparamyosin in this particular organism. However, it is present in most invertebrate species examined, including different arthropod, annelid, mollusc, and echinoderm species.

Animals↗

PubMed1995

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique.

The well-known technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects, our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue in the microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.

Animals↗

PubMed1995

Effect of motilin on the L-leucine transport in rabbit jejunum.

Motilin is a gastrointestinal peptide that stimulates the gastrointestinal motility in several species. The aim of the present work has been to determine the effect of motilin on the L-leucine absorption in rabbit jejunum. The results show that motilin inhibits the L-leucine Na(+)-dependent system of transport located in the mucosal border mainly by diminishing the apparent Vmax. Motilin did not directly affect the Na(+)-dependent system of transport, but it seems to act across the protein kinase C (PKC). These results suggest that motilin may act as a regulatory hormone of the intestinal absorption of nutrients.

Animals↗

PubMed1994

A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria.

A search of sequence data bases for a tridecamer transcription termination signal, previously described in human mtDNA as being responsible for the accumulation of mitochondrial ribosomal RNAs (rRNAs) in excess over the rest of mitochondrial genes, has revealed that this termination signal occurs in equivalent positions in a wide variety of organisms from protozoa to mammals. Due to the compact organization of the mtDNA, the tridecamer motif usually appears as part of the 3' adjacent gene sequence. Because in phylogenetically widely separated organisms the mitochondrial genome has experienced many rearrangements, it is interesting that its occurrence near the 3' end of the large rRNA is independent of the adjacent gene. The tridecamer sequence has diverged in phylogenetically widely separated organisms. Nevertheless, a well-conserved heptamer--TGGCAGA, the mitochondrial rRNA termination box--can be defined. Although extending the experimental evidence of its role as a transcription termination signal in humans will be of great interest, its evolutionary conservation strongly suggests that mitochondrial rRNA transcription termination could be a widely conserved mechanism in animals. Furthermore, the conservation of a homologous tridecamer motif in one of the last 3' secondary loops of nonmitochondrial 23S-like rRNAs suggests that the role of the sequence has changed during mitochondrial evolution.

Animals↗

PubMed1994

The complete mitochondrial DNA sequence of the crustacean Artemia franciscana.

The complete mitochondrial DNA (mtDNA) sequence of the brine shrimp Artemia franciscana has been determined. It extends the present knowledge of mitochondrial genomes to the crustacean class and supplies molecular markers for future comparative studies in this large branch of the arthropod phylum. Artemia mtDNA is 15,822 nucleotides long, and when compared with its Drosophila counterpart, it shows very few gene rearrangements, merely affecting two tRNAs placed 3' downstream of the ND 2 gene. In this position a stem-loop secondary structure with characteristics similar to the vertebrate mtDNA L-strand origin of replication is found. This suggests that, associated with tRNA changes, the diversification of the mitochondrial genome from an ancestor common to crustacea and insects could be explained by errors in the mtDNA replication process. Although the gene content is the same as in most animal mtDNAs, the sizes of the protein coding genes are in some cases considerably smaller. Artemia mtDNA uses the same genetic code as found in insects, ATN and GTG are used as initiation codons, and several genes end in incomplete T or TA codons.

Animals↗

PubMed1994

Speciation in the Artemia genus: mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps.

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the C<==>T substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.

Amino Acid Sequence↗

PubMed1994

Enhancement of the immunocytochemical detection of antigens by microwave irradiation. Benefits and limitations analysed in isolated plant nuclei and Drosophila embryos in toto.

Performing the antibody reaction under controlled heating through microwave irradiation results in significant improvements in the immunovisualization of antigens, such as shortening the times of incubation, lowering the antibody titres used and allowing the detection of difficult, inaccessible antigens. In addition to investigate the basis of the enhancement, we have extended to more intact samples such as isolated plant nuclei and in toto Drosophila embryos the results previously reported only with tissue sections. A drop of heterologous anti-nucleolin antibody covering isolated nuclei from onion root meristems spread on a glass slide was microwave irradiated, resulting in clear immunofluorescent labelling of the nucleoli. This result was never previously obtained in the absence of microwave treatment, even using the complicated procedure previously reported for the homologous identification of this nuclear protein. Using the much larger and to some extent impermeable Drosophila embryos, we were able to show that the incubation time and concentration of the anti-myosin antibody can be strongly reduced by performing the reaction at 45 degrees C under microwave irradiation. The controlled increase in temperature is the main factor responsible for these improvements; the importance of maintaining an adequate mixing of the samples is also emphasized. The proper implementation of these two experimental conditions will require the introduction of appropriate mixing accessories and temperature measuring probes for samples of small volume in current microwave laboratory ovens.

Animals↗