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Biomedical subjects

M Garcia

Publications and source records attributed to M Garcia.

At least 19 recordsLinked to original sources
PubMed2003

Synaptic process in the rat (Rattus norvegicus): Influence of methodology on results.

Synaptonemal complex (SC) analysis is a widely used method for assessing the effects of genotoxic agents in germ cells. Although the evolution of the SCs and their related annexed structures, such as nucleoli, has been well established, sometimes it is difficult to assess whether the abnormal features observed correspond to genotoxic effects or to an artefact related to the method used to obtain the SC preparations. In this article, we describe a new method of obtaining SC preparations for electron microscopy, as well as the results of a study of the first meiotic prophase in oocytes and spermatocytes of the rat (Rattus norvegicus Sprague Dawley) in which we analysed how the methodology used can influence the results. Besides important sex-specific differences, mainly during desynapsis (diplotene), a relationship between several bivalents and nucleolar structures, that in some cases could disturb the synaptic process, was observed in oocytes. At the same time, the characteristic SC fragmentation in oocytes was verified, but this fragmentation, in addition to a sex-specific component, was influenced by the method itself. By reducing to a minimum the artefacts produced by the method, it is possible to optimise the analysis of SCs as a method of testing genotoxic effects in the germ line.

Animals↗
PubMed2003

How to target estrogen receptor-negative breast cancer?

Estrogen receptor (ER)-positive breast cancers generally have a better prognosis and are often responsive to anti-estrogen therapy, which is the first example of a successful therapy targeted on a specific protein, the ER. Unfortunately ER-negative breast cancers are more aggressive and unresponsive to anti-estrogens. Other targeted therapies are thus urgently needed, based on breast cancer oncogene inhibition or suppressor gene activation as far as molecular studies have demonstrated the alteration of expression, or structure of these genes in human breast cancer. Using the MDA-MB.231 human breast cancer cell line as a model of ER-negative breast cancers, we are investigating two of these approaches in our laboratory. Our first approach was to transfect the ER or various ER-deleted variants into an ER-negative cell line in an attempt to recover anti-estrogen responsiveness. The unliganded receptor, and surprisingly estradiol, were both found to inhibit tumor growth and invasiveness in vitro and in vivo. The mechanisms of these inhibitions in ER-negative cancer cells are being studied, in an attempt to target the ER sequence responsible for such inhibition in these cancer cells. Another strategy is trying to inhibit the activity or expression of an oncogene specifically overexpressed in most breast cancers. This approach was recently shown by others to be efficient in breast cancer therapy with HER2-Neu oncogene amplification using Herceptin. Without excluding other molecular putative targets, we have focused our research on cathepsin D as a potential target, since it is often overexpressed in aggressive human breast cancers, including ER-negative tumors, and rarely associated with HER2-Neu amplification. Our first results obtained in vitro on cell lines and in vivo in tumor xenografts in nude mice, illustrate that the mode of action of cathepsin D in breast cancer is useful to guide the development of these therapies. In the past 20 years we have learned that the action of cathepsin D is complex and involves both intracellular and extracellular activities due to its proteolytic activity and to interactions with membrane components without catalytic activity. Each of these mechanisms could be potentially inhibited in an attempt to prevent tumor growth. Breast cancer is a very heterogeneous and multigenic disease and different targeted therapies adapted to each category of breast cancer are therefore required. Validated assays in the primary tumor of molecular markers such as ER, HER2-Neu and cathepsin D should help to predict which targeted therapy should be applied to cure breast cancer patients.

Breast Neoplasms↗
PubMed2003

Factors associated with time to sputum smear conversion in active pulmonary tuberculosis.

OBJECTIVE: To ascertain the factors affecting the time between the initiation of treatment and obtaining three negative sputum smears. DESIGN: In a study of 109 patients with pulmonary tuberculosis, the main variable was the period during which the patients had sputum smears once treatment was initiated. Multivariate analysis (multiple linear regression) was performed to document those variables independently associated with time to conversion. RESULTS: The patients had positive smears for a mean of 28.63 days. The most frequent radiographic pattern was cavitary disease (36.7%). HIV co-infection was present in 38.5% of the patients. HIV-infected patients showed a cavitation pattern in only 9.6% vs 52.2% of patients without HIV infection (P < 0.001). The variables that showed a statistically significant and independent relationship with the time to sputum smear conversion were pulmonary radiographic pattern, age and erythrocyte sedimentation rate (ESR). CONCLUSIONS: ESR, age and the presence of cavitary disease seem to be factors associated with a longer time to sputum smear conversion in patients with active pulmonary tuberculosis. However, HIV co-infection is associated with a shorter time to sputum conversion. A key factor is therefore the presence or not of cavitation, independently of HIV infection.

Adult↗
PubMed2002

Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA.

BACKGROUND: The mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder of early childhood characterized by decreased mtDNA copy number in affected tissues. Recently, MDS has been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxy-guanosine kinase (dGK). Mutations in TK2 have been associated with the myopathic form of MDS, and mutations in dGK with the hepatoencephalopathic form. OBJECTIVES: To further characterize the frequency and clinical spectrum of these mutations, the authors screened 20 patients with myopathic MDS. RESULTS: No patient had dGK gene mutations, but four patients from two families had TK2 mutations. Two siblings were compound heterozygous for a previously reported H90N mutation and a novel T77M mutation. The other siblings harbored a homozygous I22M mutation, and one of them had evidence of lower motor neuron disease. The pathogenicity of these mutations was confirmed by reduced TK2 activity in muscle (28% to 37% of controls). CONCLUSIONS: These results show that the clinical expression of TK2 mutations is not limited to myopathy and that the myopathic form of MDS is genetically heterogeneous.

Child, Preschool↗
PubMed2002

Distribution of intrachromosomal telomeric sequences (ITS) on Macaca fascicularis (Primates) chromosomes and their implication for chromosome evolution.

The intrachromosomal location of the telomeric sequence in the crab-eating macaque, Macaca fascicularis (F. Cercopithecidae, Catarrhini) has been analysed by fluorescent in situ hybridisation with a long synthetic (TTAGGG)(n) probe. A total of 237 metaphases was analysed. As expected, all telomeres hybridised with the probe and 90 intrachromosomal loci with different hybridisation frequencies were also detected. The chromosomal location of interstitial telomeric sequences in M. fascicularis and in Homo sapiens was then compared, 37 sites (41.11%) being found to be conserved. Some of these sequences can be derived from rearrangements, such as inversions (MFA13q23) or fusions (MFA2p13 and MFA13p12), that have taken place during karyotype evolution.

Animals↗
PubMed2002

Chromosomal homologies between Cebus and Ateles (primates) based on ZOO-FISH and G-banding comparisons.

ZOO-FISH (Fluorescent "in vitro" hybridization) was used to establish the chromosomal homology between humans (HSA) and Cebus nigrivitatus (CNI) and Ateles belzebuth hybridus (ABH). These two species belong to different New World monkey families (Cebidae and Atelidae, respectively) which differ greatly in chromosome number and in chromosome morphology. The molecular results were followed by a detailed banding analysis. The ancestral karyotype of Cebus was then determined by a comparison of in situ hybridization results, as well as chromosomal morphology and banding in other Platyrrhini species. The karyotypes of the four species belonging to the genus Cebus differ from each other by three inversions and one fusion as well as in the location and amounts of heterochromatin. Results obtained by ZOO-FISH in ABH are in general agreement with previous gene-mapping and in situ hybridization data in Ateles, which show that spider monkeys have highly derived genomes. The chromosomal rearrangements detected between HSA and ABH on a band-to-band basis were 27 fusions/fissions, 12 centromeric shifts, and six pericentric inversions. The ancestral karyotype of Cebus was then compared with that of Ateles. The rearrangements detected were 20 fusions/fissions, nine centromeric shifts, and five inversions. Atelidae species are linked by a fragmentation of chromosome 4 into three segments forming an association of 4/15, while Ateles species are linked by 13 derived associations. The results also helped clarify the content of the ancestral platyrrhine karyotype and the mode of chromosomal evolution in these primates. In particular, associations 2/16 and 5/7 should be included in the ancestral karyotype of New World monkeys.

Animals↗
PubMed2002

Dynamics and stability of legged locomotion in the horizontal plane: a test case using insects.

Motivated by experimental studies of insects, we propose a model for legged locomotion in the horizontal plane. A three-degree-of freedom, energetically conservative, rigid-body model with a pair of compliant virtual legs in intermittent contact with the ground allows us to study how dynamics depends on parameters such as mass, moment of inertia, leg stiffness, and length. We find periodic gaits, and show that mechanics alone can confer asymptotic stability of relative heading and body angular velocity. We discuss the relevance of our idealized models to experiments and simulations on insect running, showing that their gait and force characteristics match observations reasonably well. We perform parameter studies and suggest that our model is relevant to the understanding of locomotion dynamics across species.

Animals↗
PubMed2002

Attention-deficit/hyperactivity disorder (ADHD): feasibility of linkage analysis in a genetic isolate using extended and multigenerational pedigrees.

Segregation analyses converge in explaining the predisposition to attention-deficit/hyperactivity disorder (ADHD) as the consequence of a major gene and exclude purely environmental or cultural transmission. As a result of the ADHD phenotype restrictions, collection of extended families or design of linkage studies using families has been extremely difficult and thus currently linkage studies have been performed using only concordant or discordant sib-pairs rather than large families. On the other hand, intergenerational studies are represented by the transmission disequilibrium test (TDT) using trios. We collected pedigree data on ADHD from the Paisa community from Antioquia, Colombia, a genetic isolate. The goal of this study was to genetically map a putative gene predisposing to ADHD in a set of 27 multigenerational Paisa families. Here we present the results of a power simulation using SIMLINK to detect linkage of ADHD. ADHD was assumed to be a dichotomous trait with incomplete penetrance and a phenocopy rate of 3% in males and 0.2% in females. We simulated cosegregation of the trait and a marker locus in our pedigrees. We assumed Hardy-Weinberg and linkage equilibrium, equally frequent marker alleles and evaluated power at several recombination fractions between the trait and marker loci. Also, the ADHD trait was assumed to be genetically heterogeneous and different functions of age-dependent penetrance were simulated. We found exceptionally good power to detect linkage (expected LOD > 14 if theta is 0.1 or less), and that the presence of heterogeneity up to 50% does not affect substantially the projected LOD scores even for a theta recombination value of 0.05 (eLOD > 5.87). Having now obtained blood samples and confirmatory interviews in five families (representing 20% of the projected number of families), we performed a new analysis. The expected mean LOD in these five families reached values close to 10 and remained invariant when heterogeneity and different penetrance models were considered. We discuss the relative benefits of using extended and multigenerational families for genetic mapping studies as opposed to using nuclear families, affected sib pairs or sporadic cases which require the collection of over 1000 analytical units to get the same power exhibited by the small number of pedigrees described here.

Adult↗
PubMed2002

Air reduction of intussusception.

AIM: To show that intussusception in children is reducible in most cases, using air. METHODS: Initially we used a barium enema to treat paediatric intussusception. Now we use an air enema. We consider the latter reduction technique to be the method of choice in the treatment of intussusception in childhood. RESULTS: Over a period of 21 years, we have treated 151 patients presenting with intussusception. In a group of 130 children treated with barium enema we managed to reduce the intussusception in 110 cases (84.6 %). We used air pressure to treat a second group of 21 children; the success rate in this group was 100 %. CONCLUSIONS: In order to avoid a surgical operation this technique must become more generally known. We are of the opinion that if the method is applied correctly, it is almost always possible to correct intussusception using air.

Air↗
PubMed2002

Is Doppler tissue velocity during early left ventricular filling preload independent?

BACKGROUND: Transmitral Doppler flow indices are used to evaluate diastolic function. Recently, velocities measured by Doppler tissue imaging have been used as an index of left ventricular relaxation. OBJECTIVE: To determine whether Doppler tissue velocities are influenced by alterations in preload. METHODS: Left ventricular preload was altered in 17 patients (all men, mean (SD) age, 49 (8) years) during echocardiographic measurements of left ventricular end diastolic volume, maximum left atrial area, peak early Doppler filling velocity, and left ventricular myocardial velocities during early filling. Preload altering manoeuvres included Trendelenberg (stage 1), reverse Trendelenberg (stage 2), and amyl nitrate (stage 3). Systolic blood pressure was measured at each stage. RESULTS: In comparison with baseline, left ventricular end diastolic volume (p = 0.001), left atrial area (p = 0.003), peak early mitral Doppler filling velocity (p = 0.01), and systolic blood pressures (p = 0.001) were all changed by preload altering manoeuvres. Only left ventricular myocardial velocity during early filling remained unchanged by these manoeuvres. CONCLUSIONS: In contrast to standard transmitral Doppler filling indices, Doppler tissue early diastolic velocities are not significantly affected by physiological manoeuvres that alter preload. Thus Doppler tissue velocities during early left ventricular diastole may provide a better index of diastolic function in cardiac patients by providing a preload independent assessment of left ventricular filling.

Blood Flow Velocity↗
PubMed2002

The efficacy of chloroquine for the treatment of acute, uncomplicated, Plasmodium falciparum malaria in Laos.

To assess the local efficacy of chloroquine for the treatment of acute, uncomplicated, Plasmodium falciparum malaria, children and adults from Sekong province (an area of Laos with a low intensity of transmission) were tested in a 28-day, in-vivo study. Complete data were collected from 88 of the 102 subjects enrolled between October 1999 and September 2000. After genotypic analysis to distinguish recrudescing infections from re-infections, 35 (39.7%, with a 95% confidence interval of 29.5%-50.7%) of these 88 patients were considered treatment failures. These results seriously question the use of chloroquine as the first-line treatment for P. falciparum malaria in the study area.

Acute Disease↗
PubMed2001

A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells.

Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its over-expression by transfection stimulates cancer cell proliferation. The mechanism by which this protease affects proliferation remains, however, unknown. In order to determine whether proteolytic activity is necessary, we abolished its enzymatic activity using site-directed mutagenesis followed by stable transfection in 3Y1-Ad12 cancer cells. Substitution of the aspartic acid residue 231 by an asparagine residue in its catalytic site abrogated the cathepsin-D proteolytic activity but did not affect its expression level, processing or secretion. However, like wild-type cathepsin-D, this mutated catalytically-inactive cathepsin-D retained its capacity to stimulate proliferation of cells embedded in Matrigel or collagen I matrices, colony formation in soft agar and tumor growth in athymic nude mice. Addition on the mock-transfected cells, of either conditioned media containing the wild-type or the mutated pro-cathepsin-D, or of the purified mutated pro-cathepsin-D, partially mimicked the mitogenic activity of the transfected cathepsin-D, indicating a role of the secreted pro-enzyme. Moreover, addition of two anti-cathepsin-D antibodies on the cathepsin-D transfected cells inhibited their proliferation, suggesting an action of the secreted pro-cathepsin-D via an autocrine loop. A synthetic peptide containing the 27-44 residue moiety of the cathepsin-D pro-fragment was, however, not mitogenic suggesting that a receptor for the pro-fragment was not involved. Furthermore, the cathepsin-D mitogenicity was not blocked by inhibiting the interaction of pro-cathepsin-D with the mannose-6-phosphate receptors. Our results altogether demonstrate that a mutated cathepsin-D devoid of catalytic activity is still mitogenic and suggest that it is acting extra-cellularly by triggering directly or indirectly a yet unidentified cell surface receptor.

Animals↗
PubMed2001

Cembranoid and long-chain alkanol sites on the nicotinic acetylcholine receptor and their allosteric interaction.

Long-chain alkanols are general anesthetics which can also act as uncharged noncompetitive inhibitors of the peripheral nicotinic acetylcholine receptor (AChR) by binding to one or more specific sites on the AChR. Cembranoids are naturally occurring, uncharged noncompetitive inhibitors of peripheral and neuronal AChRs, which have no demonstrable general anesthetic activity in vivo. In this study, [3H]tenocyclidine ([3H]TCP), an analogue of the cationic noncompetitive inhibitor phencyclidine (PCP), was used to characterize the cembranoid and long-chain alkanol sites on the desensitized Torpedo californica AChR and to investigate if these sites interact. These studies confirm that there is a single cembranoid site which sterically overlaps the [3H]TCP channel site. This cembranoid site probably also overlaps the sites for the cationic noncompetitive inhibitors, procaine and quinacrine. Evidence is also presented for one or more allosteric cembranoid sites which negatively modulate cembranoid affinity for the inhibitory site. In contrast, long-chain alkanols inhibit [3H]TCP binding through an allosteric mechanism involving two or more alkanol sites which display positive cooperativity toward each other. Double inhibitor studies show that the cembranoid inhibitory site and the alkanol sites are not independent of each other but interfere allosterically with each other's inhibition of [3H]TCP binding. The simplest models consistent with the observed data are presented and discussed.

Allosteric Regulation↗
PubMed2001

Prediction of stable walking for a toy that cannot stand.

Previous experiments [M. J. Coleman and A. Ruina, Phys. Rev. Lett. 80, 3658 (1998)] showed that a gravity-powered toy with no control and that has no statically stable near-standing configurations can walk stably. We show here that a simple rigid-body statically unstable mathematical model based loosely on the physical toy can predict stable limit-cycle walking motions. These calculations add to the repertoire of rigid-body mechanism behaviors as well as further implicating passive dynamics as a possible contributor to stability of animal motions.

Animals↗
PubMed2001

Porcine neonatal pancreatic cell clusters in tissue culture: benefits of serum and immobilization in alginate hydrogel.

Porcine neonatal pancreatic cell clusters (NPCCs) may be a suitable source of insulin producing tissue for transplantation in diabetic patients. The possible beneficial effect of serum on maturation of NPCCs in vitro is difficult to achieve because of cell clumping, which can be avoided by immobilization in alginate hydrogel matrix. Collagenase treated pancreata, cultured for 4 days, formed NPCCs that were embedded in alginate cross-linked with CaCl2 and cultured in modified Ham's F10 medium with 10% fetal calf serum (FCS) for 10 days. NPCCs cultured as suspension in F10+ with 0.5% bovine serum albumin or with 10% FCS were used as control. To prevent the aggregation when cultured with serum, NPCCs were kept as a very diluted suspension. At the beginning and end of the culture, samples were taken for insulin and DNA content and immunostained for beta and non-beta cells. The culture of NPCCs immobilized in alginate resulted with 3-fold increase in insulin content and 9-fold increase in insulin/DNA ratio. Histology revealed evident increase of number of insulin- and other hormone-positive cells compared with the control. Even though 2 weeks in culture resulted in impaired glucose-induced insulin release, the amount of insulin secreted by clusters cultured in the presence of serum was 4-fold higher than in serum-free conditions. After transplantation, NPCCs retrieved from alginate reversed hyperglycemia similarly to NPCCs cultured in standard conditions. In conclusion, this study shows the feasibility of in vitro immobilization of NPCCs in alginate three-dimensional matrix, allowing cell clusters to be cultured at least two times higher density compared with culture in suspension.

Alginates↗