Biomedical subjects

A Rojas

Publications and source records attributed to A Rojas.

At least 37 records · Page 2Linked to original sources

PubMed2001

Albumin-derived advanced glycation end-products trigger the disruption of the vascular endothelial cadherin complex in cultured human and murine endothelial cells.

Endothelial cell (EC) junctions regulate in large part the integrity and barrier function of the vascular endothelium. Advanced glycation end-products (AGEs), the irreversibly formed reactive derivatives of non-enzymic glucose-protein condensation reactions, are strongly implicated in endothelial dysfunction that distinguishes diabetes- and aging-associated vascular complications. The aim of the present study was to determine whether AGEs affect EC lateral junction proteins, with particular regard to the vascular endothelial cadherin (VE-cadherin) complex. Our results indicate that AGE-modified BSA (AGE-BSA), a prototype of advanced glycated proteins, disrupts the VE-cadherin complex when administered to ECs. AGE-BSA, but not unmodified BSA, was found to induce decreases in the levels of VE-cadherin, beta-catenin and gamma-catenin in the complex and in total cell extracts, as well as a marked reduction in the amount of VE-cadherin present at the cell surface. In contrast, the level of platelet endothelial cell adhesion molecule-1 (PECAM-1), which is located at lateral junctions, was not altered. Supplementation of the cellular antioxidative defences abolished these effects. Finally, the loss of components of the VE-cadherin complex was correlated with increases in vascular permeability and in EC migration. These findings suggest that some of the AGE-induced biological effects on the endothelium could be mediated, at least in part, by the weakening of intercellular contacts caused by decreases in the amount of VE-cadherin present.

Acetylcysteine↗

PubMed2001

Structural and functional interactions of transcription factor (TF) IIA with TFIIE and TFIIF in transcription initiation by RNA polymerase II.

A topological model for transcription initiation by RNA polymerase II (RNAPII) has recently been proposed. This model stipulates that wrapping of the promoter DNA around RNAPII and the general initiation factors TBP, TFIIB, TFIIE, TFIIF and TFIIH induces a torsional strain in the DNA double helix that facilitates strand separation and open complex formation. In this report, we show that TFIIA, a factor previously shown to both stimulate basal transcription and have co-activator functions, is located near the cross-point of the DNA loop where it can interact with TBP, TFIIE56, TFIIE34, and the RNAPII-associated protein (RAP) 74. In addition, we demonstrate that TFIIA can stimulate basal transcription by stimulating the functions of both TFIIE34 and RAP74 during the initiation step of the transcription reaction. These results provide novel insights into mechanisms of TFIIA function.

Animals↗

PubMed2001

Requirement of multiple protein domains and residues for gating K(ATP) channels by intracellular pH.

ATP-sensitive K(+) channels (K(ATP)) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K(ATP) by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating K(ATP) by protons are demonstrated. The pH sensitivity enables the K(ATP) to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.

Adenosine Triphosphate↗

PubMed2001

Microencapsulation of an anti-VE-cadherin antibody secreting 1B5 hybridoma cells.

Accumulating experimental evidence demonstrates that tumor growth and lethality are dependent on angiogenesis. Based on this concept, there is growing interest in the use of antiangiogenesis agents to inhibit tumor expansion. Compelling data implicate vascular endothelium (VE)-cadherin (an endothelium specific protein) as a key factor in the last step of angiogenesis, where the endothelial cells join one to each other and form microtubules (future blood vessels). We propose a novel approach to the inhibition of angiogenesis by immobilizing VE-cadherin-secreting hybridoma cells in alginate-agarose microcapsules. Hybridoma cells can be protected with biocompatible and semipermeable membranes that permit exit of anti-VE-cadherin monoclonal antibodies but not entry of cellular immune mediators. Stability studies were performed to select the suitable microcapsule for cell immobilization. Alginate and agarose solid beads coated with poly-L-lysine and alginate were chosen according to their stability and diffusional properties. 1B5 hybridoma cells were grown within the microcapsules and secreted anti-VE-cadherin antibodies during the 9 days of culture, reaching a cumulative concentration of 1.7 microg/mL. This antibody concentration inhibited microtubule formation (87%) in the in vitro angiogenesis Matrigel assay. Moreover, the antiangiogenic effect observed was antibody concentration related. These findings open a new alternative for the inhibition or prevention of angiogenesis and demonstrates the feasibility of using microencapsulated cells as a control-drug delivery system.

Alginates↗

PubMed2001

Modulation of the heteromeric Kir4.1-Kir5.1 channels by P(CO(2)) at physiological levels.

Several inward rectifier K(+) (Kir) channels are pH-sensitive, making them potential candidates for CO(2) chemoreception in cells. However, there is no evidence showing that Kir channels change their activity at near physiological level of P(CO(2)), as most previous studies were done using high concentrations of CO(2). It is known that the heteromeric Kir4.1-Kir5.1 channels are highly sensitive to intracellular protons with pKa value right at the physiological pH level. Such a pKa value may allow these channels to regulate membrane potentials with modest changes in P(CO(2)). To test this hypothesis, we studied the Kir4.1-Kir5.1 currents expressed in Xenopus oocytes and membrane potentials in the presence and absence of bicarbonate. Evident inhibition of these currents (by approximately 5%) was seen with P(CO(2)) as low as 8 torr. Higher P(CO(2)) levels (23-60 torr) produced stronger inhibitions (by 30-40%). The inhibitions led to graded depolarizations (5-45 mV with P(CO(2)) 8-60 torr). Similar effects were observed in the presence of 24 mM bicarbonate and 5% CO(2). Indeed, the Kir4.1-Kir5.1 currents were enhanced with 3% CO(2) and suppressed with 8% CO(2) in voltage clamp, resulting in hyper- (-9 mV) and depolarization (16 mV) in current clamp, respectively. With physiological concentration of extracellular K(+), the Kir4.1-Kir5.1 channels conduct substantial outward currents that were similarly inhibited by CO(2) as their inward rectifying currents. These results therefore indicate that the heteromeric Kir4.1-Kir5.1 channels are modulated by a modest change in P(CO(2)) levels. Such a modulation alters cellular excitability, and enables the cell to detect hypercapnia and hypocapnia in the presence of bicarbonate.

Animals↗

PubMed2001

Effects of phycocyanin extract on tumor necrosis factor-alpha and nitrite levels in serum of mice treated with endotoxin.

Phycocyanin is a biliprotein which exerts antioxidative and anti-inflammatory effects in various in vivo and in vitro experimental models. In this study phycocyanin effects on tumor necrosis factor-alpha (TNF alpha) and nitrite levels in serum of mice treated with lipopolysaccharide (LPS) was examined. TNF alpha was measured by cytotoxicity on L-929 cells and nitrite by the Griess reaction, after reduction of all nitrates to nitrites by nitrate reductase, 1 h after LPS injection (0.5 mg/kg i.p.) there was a significant increase in TNF alpha levels in mouse serum. Phycocyanin (50-300 mg/kg p.o.), administered 1 h before LPS, reduced dose-dependently the TNF alpha concentration in serum. After 18 h, LPS (30 mg/kg i.p.) also induced a substantial increase in serum nitrite levels, which were reduced dose-dependently by phycocyanin pretreatment (100-300 mg/kg p.o.). The results indicate that phycocyanin exerts inhibitory effects on TNF alpha and NO production which might be ascribed to the antioxidative properties of the biliprotein.

Animals↗

PubMed2001

Treatment of Trypanosoma cruzi-infected children with nifurtimox: a 3 year follow-up by PCR.

Patients suffering from Chagas' disease, as determined by positive serological results, were tested for further evidence of Trypanosoma cruzi infection by xenodiagnosis and PCR. The patients included 67 children aged from 0 to 10 years and 75 adults. All children were positive by PCR on their pre-therapy sample, while only 69% of the seropositive adults and none of the 78 seronegative control adults were PCR positive. Xenodiagnosis was positive in 79% of the children, but only in 21% of the adults. A group of 66 children was treated with nifurtimox, and followed up every 3 months during the first year and every 6 months during the second and third year post-therapy, by PCR, xenodiagnosis and serology. We concluded that PCR was the most effective test to monitor children for 3 years post-chemotherapy, when all the cases converted from positive to negative. Conventional serology, however, remained positive after that period in most cases. In contrast, conversion to negative xenodiagnosis occurred very early after treatment.

Adult↗

PubMed2001

Synergism between Phyllobacterium sp. (N(2)-fixer) and Bacillus licheniformis (P-solubilizer), both from a semiarid mangrove rhizosphere.

Mangrove seedlings were treated with a mixture of two bacterial species, the slow-growing, N(2)-fixing bacterium Phyllobacterium sp. and the fast-growing, phosphate-solubilizing bacterium Bacillus licheniformis, both isolated from the rhizosphere from black, white, and red mangroves of a semiarid zone. Nitrogen fixation and phosphate solubilization increased when the mixture was used compared to the effects observed when adding individual cultures, notwithstanding that there was no increase in bacterial multiplication under these conditions. Inoculation of black mangrove seedlings in artificial seawater showed the mixture performed somewhat better than inoculation of the individual bacterium; more leaves were developed and higher levels of (15)N were incorporated into the leaves, although the total nitrogen level decreased. This study demonstrates that interactions between individual components of the rhizosphere of mangroves should be considered when evaluating these bacteria as plant growth promoters.

Journal Article↗

PubMed2001

Differential interleukin-8 response of intestinal epithelial cell line to reactogenic and nonreactogenic candidate vaccine strains of Vibrio cholerae.

In this study, we analyzed whether attachment of Vibrio cholerae vaccine strains to human intestinal epithelial cells can induce an interleukin-8 (IL-8) response. The IL-8 transcripts were detected by PCR amplification of reverse-transcribed mRNA, and the gene product secretion was measured by an enzyme-linked immunosorbent assay. Infection of monolayers of the undifferentiated HT29-18N2 cell line with reactogenic (JBK70 and 81) and nonreactogenic (CVD103HgR and 638) vaccine strains of V. cholerae resulted in markedly higher IL-8 expression by epithelial cells exposed to reactogenic strains than by cells exposed to the nonreactogenic strains. Additionally, epithelial cells produced IL-8 transcripts following stimulation with cholera vaccine strains in a concentration-dependent manner. These results represent a new insight into the inflammatory component of reactogenicity and could be used as a predictive marker of vaccine reactogenicity prior to human testing.

Bacterial Adhesion↗

PubMed2001

Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E.

In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.

Anti-Bacterial Agents↗

PubMed2001

Green fluorescent protein-tagged Leishmania in phlebotomine sand flies.

In this work we have used for the first time green fluorescent protein (GFP) tagged cells of the human parasite Leishmania donovani to observe its development in the gut of phlebotomine sand flies. Low numbers of GFP-tagged L. donovani were more easily detected than nontagged Leishmania, suggesting that GFP-tagged Leishmania could be used to efficiently study the biology of Leishmania in their vectors, and open the possibility of using nonaxenic flies. Using this method, we found that GFP-tagged L. donovani, the ethiological agent of Old World Kala-azar, were able to establish an infection within the gut of Lutzomyia species, which are vectors of New World Leishmania. The GFP-tagged parasites divide successfully in the gut of colonized and in wild caught Lu. longipalpis (Lutz & Neiva, 1912), Lu. ovallesis (Ortiz, 1952), and Lu. youngi (Feliciangeli & Murillo, 1985). In the case of Lulongipalpis the labeled parasite exhibited a normal anterior development as the one observed in its natural vector.

Animals↗

PubMed2001

Detection and significance of inapparent infection in Chagas disease in western Venezuela.

Inapparent infections of Trypanosoma cruzi were detected in symptomless seropositive people living in close proximity, and under the same conditions of risk, to patients with acute Chagas disease. Similar infections were also detected in sera samples of people from 25 villages of western Venezuela where Chagas disease is endemic. Seropositivity in all the 1,251 studied samples was established by use of 3 serological methods (direct agglutination test, indirect immunofluorescence antibody test, and enzyme-linked immunosorbent assay). Each seropositive sample was tested for detection of anti-T. cruzi-specific immunoglobulin (Ig) M and IgG levels and specific T. cruzi infection by molecular methodology (polymerase chain reaction assay). The combined analysis of the serologic (IgM and IgG levels), molecular (specific T. cruzi DNA), and statistical findings demonstrated the existence of a different stage of T. cruzi infection in asymptomatic patients, which is suggested to be recognized as inapparent infection. Its definition, significance, and comparison with typical Chagas disease phases are presented, and its potential epidemiological importance is discussed.

Adolescent↗

PubMed2001

Detection of Leishmania causing visceral leishmaniasis in the Old and New Worlds by a polymerase chain reaction assay based on telomeric sequences.

We present a new polymerase chain reaction assay based on telomeric sequences of Leishmania donovani. When this assay was used in dilutions of purified L. donovani DNA, a strong amplification signal was observed with 1 fg of DNA. In a specificity test that used purified DNA from Old World and New World Leishmania, the assay recognized all parasites isolated from patients with visceral leishmaniasis, except for 2 isolates of Leishmania colombiensis from Venezuela and 1 isolate from Brazil. All Leishmania major and Leishmania tropica isolates tested were negative, except for one isolate in each species. We also used the assay on fresh and archive bone marrow samples recovered from Giemsa-stained slides and from dried blood stains.

Adult↗

PubMed2001

Evaluation of a Triatoma infestans elimination program by the decrease of Trypanosoma cruzi infection frequency in children younger than 10 years, Chile, 1991-1998.

Chagas disease is widespread in Chile, distributed in rural and periurban areas in the 7 most northern regions of the country. The principal vector of Trypanosoma cruzi is Triatoma infestans. The interruption of the domestic cycle of transmission of T. cruzi has been attempted by health education, human housing improving, and elimination of the vector by means of systematic insecticide spraying of human dwellings. Spraying with insecticides has been supported by Chile's health authorities and has been carried out for the last 12 years. A total of 13,280 children (aged up to 10 years) were randomly selected from 47 counties in the area of Chile endemic for Chagas disease, and blood samples were collected to determine the levels of antibodies to T. cruzi by indirect hemagglutination and enzyme-linked immunosorbent assay tests. The results of the tests were analyzed to determine the changes that occurred after 12 years of insecticide spraying of dwellings to eliminate T. infestans infestation. A total of 142 (1.1%) samples of children showed antibodies to T. cruzi. This rate is significantly lower than the data generated in similar studies conducted in 1982-1985. The following reduction in prevalence rates were observed in each of the 7 endemic regions of the country: region I, 5.5-0.3%; region II, 6.6-0.3%; region III, 9.8-1.0%; region IV, 7.2-2.0%; region V, 5.2-1.9%, Metropolitan region, 1.4-0.6%; and region VI, 1.4-0.4%. Serovigilance of T. cruzi antibodies level represents a novel approach that may allow the evaluation of the impact of the vector elimination program. The results identify regions that need to strengthen the efforts to reduce the insect infestation of dwellings.

Animals↗

PubMed2001

[High prevalence of subclinical thyroidal disease among individuals attended in health control].

BACKGROUND: There is no information about the prevalence of thyroidal diseases in the general Chilean population. AIM: To assess the prevalence of thyroidal diseases in individuals attended in occupational health examinations. SUBJECTS AND METHODS: Four hundred seventy two individuals were examined between 1998 and 1999. In all, serum levels of thyroid hormones, TSH and anti thyroidal antibodies (anti microsomal, anti thyroid peroxidase and anti thyroglobulin) were measured. RESULTS: Forty four subjects were excluded from the study due to an incomplete medical record and 18 due to a personal history of thyroidal disease. Abnormal serum levels of thyroid hormones or TSH were detected in 28 subjects (6.8%). Four (1%) had hypothyroidism, 23 a subclinical hypothyroidism (5.6%) and one (0.2%) had hyperthyroidism. In 87 subjects (21.2%) at least one of the antibodies was positive. Positive anti thyroid antibodies were found in 14 of 28 subjects (50%) with abnormal thyroid hormone levels, compared with 73 of 382 subjects (19.1%) with normal thyroid hormone levels. Thyroid dysfunction was twice as frequent in women than in men. CONCLUSIONS: In this sample, a 6.8% prevalence of abnormal thyroid function tests was detected.

Adolescent↗

PubMed2000

Regulation of endothelial nitric oxide synthase expression by albumin-derived advanced glycosylation end products.

We examined whether albumin-derived advanced glycosylation end products (AGEs) downregulate the expression of endothelial nitric oxide synthase (NOS). Significant reductions in NOS activity and cGMP levels in bovine aortic endothelial cells were observed when exposed to different concentrations of albumin-derived AGEs. Western and Northern blot analyses showed significant decreases at the protein and transcript levels. Both reductions became evident after 24 hours of exposure. Nuclear run-on assays showed that AGE-BSA did not modify the transcription rate of the NOS III gene; however, AGE-BSA treatment markedly reduced the half-life of NOS III mRNA. In addition, AGE-treated endothelial cells displayed significant reduction on their antiplatelet properties. These results indicate that NOS expression is reduced by AGEs by increasing the rate of mRNA degradation and may be relevant to the impairment of some endothelial functions observed in diabetes and aging. The full text of this article is available at http://www.circresaha.org.

Animals↗

PubMed2000

ARCON: accelerated radiotherapy with carbogen and nicotinamide in head and neck squamous cell carcinomas. The experience of the Co-operative group of radiotherapy of the european organization for research and treatment of cancer (EORTC).

BACKGROUND: Since there is increasing evidence that both acute (perfusion-limited) and chronic (diffusion-limited) hypoxia, and tumor repopulation may prejudice the outcome of radiotherapy, the combination of carbogen (95% oxygen-5% carbon dioxide) and nicotinamide with accelerated radiotherapy (ARCON) should reduce the impact of these factors of radioresistance. AIM: This clinical study was aimed at determining the feasibility, as well as the qualitative and quantitative toxic effects of a therapeutic approach based on ARCON, and assessing the tumor response rates that can be achieved with this regime in patients with locally advanced tumors of the head and neck. METHODS: A phase I/II study conducted between 1993 and 1996 by the Co-operative Group of Radiotherapy of the EORTC included three consecutive steps: accelerated fractionation (AF) combined with carbogen (11 analyzable patients), AF combined with the daily administration of nicotinamide (n=10), and AF with both carbogen and nicotinamide (n=17). Radiotherapy was based on an accelerated regime (72 Gy in 5.5 weeks). Nicotinamide was delivered 90 min before the first irradiation session, at a daily dose of 6 g. Carbogen breathing started 5 min before irradiation and lasted throughout the entire radiotherapy sessions. RESULTS: No significant difference in loco-regional toxicity was found among the three study steps, when carbogen and nicotinamide, either alone or in combination, were combined with AF. The feasibility of the ARCON protocol, as proposed in the present EORTC study, appears to be significantly impaired when nicotinamide is added, at a daily dose of 6 g, to AF and carbogen, in an unselected group of patients. More than 20% of patients experienced grade 2 or 3 emesis. It also demonstrates, in unselected groups of patients, no significant difference in tumor response and local control when carbogen and nicotinamide, either alone or in combination, are added to accelerated radiotherapy. The percentages of objective response at 2 months were 81, 70 and 87%, respectively. CONCLUSION: Future ARCON trials should target selected head and neck tumor localizations and stages, and a lower nicotinamide dose is needed to reduce severe upper gastro-intestinal toxicity.

Adult↗

PubMed2000

Smooth muscle relaxing activity of gentiopicroside isolated from Gentiana spathacea.

Bioassay directed fractionation of the (1:1) chloroform-methanol extract of Gentiana spathacea H.B.K (Gentianaceae) led to the isolation of gentiopicroside (gentiopricrin) (1), the major spasmolytic component of the plant. Gentiopicroside inhibited, in a concentration-dependent manner, the spontaneous contractions of isolated guinea pig ileum. Contractions induced by histamine, acetylcholine, BaCl2 and KCl on the ileum were also significantly blocked by this monoterpene glucoside, which suggests that this compound might be interfering with calcium influx into the smooth muscle cells.

Animals↗