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Biomedical subjects

R Ros

Publications and source records attributed to R Ros.

At least 19 recordsLinked to original sources
PubMed2006

Analysis of subcellular surface structure, function and dynamics.

Analytics of single biological cells allows quantitative investigation from a structural, functional and dynamical point of view and opens novel possibilities to an unamplified subcellular analysis. In this article, we report on three different experimental methods and their applications to single cellular systems with a subcellular sensitivity down to the single molecule level. First, the subcellular surface structure of living bacteria (Corynebacterium glutamicum) was investigated with atomic force microscopy (AFM) at the resolution of individual surface layer (S-layer) proteins; discrimination of bacterial strains that lack the expression of hexagonally packed surface layer proteins was possible. Second, quantitative measurement of individual recognition events of membrane-bound receptors on living B-cells was achieved in single cell manipulation and probing experiments with optical tweezers (OT) force spectroscopy. And third, intracellular dynamics of translocating photoactivatable GFP in plant protoplasts (Nicotiana tabacum BY-2) was quantitatively monitored by two-photon laser scanning microscopy (2PLSM).

Animals↗
PubMed2006

Theoretical analysis of single-molecule force spectroscopy experiments: heterogeneity of chemical bonds.

We show that the standard theoretical framework in single-molecule force spectroscopy has to be extended to consistently describe the experimental findings. The basic amendment is to take into account heterogeneity of the chemical bonds via random variations of the force-dependent dissociation rates. This results in a very good agreement between theory and rupture data from several different experiments.

Biomechanical Phenomena↗
PubMed2004

Theoretical analysis of dynamic force spectroscopy experiments on ligand-receptor complexes.

The forced rupture of single chemical bonds in biomolecular compounds (e.g. ligand-receptor systems) as observed in dynamic force spectroscopy experiments is addressed. Under the assumption that the probability of bond rupture depends only on the instantaneously acting force, a data collapse onto a single master curve is predicted. For rupture data obtained experimentally by dynamic AFM force spectroscopy of a ligand-receptor bond between a DNA and a regulatory protein we do not find such a collapse. We conclude that the above mentioned, generally accepted assumption is not satisfied and we discuss possible explanations.

Binding Sites↗
PubMed2004

Large-scale homogeneous molecular templates for femtosecond time-resolved studies of the guest-host interaction.

Self-assembled monolayer films based on iodobenzoyloxy-functionalized resorc[4]arenes were prepared on gold substrates to serve as model systems for future time-resolved studies of molecular recognition, a mechanism of outstanding importance in bioorganic systems. The film properties were tested using X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and imaging ellipsometry. An apparatus for time-resolved electron spectroscopy utilizing femtosecond soft X-ray pulses is capable of detecting iodine core-level photolines and the photoinduced dissociation after ultraviolet illumination. The developed technique holds promise for tracking the temporal evolution of chemical shifts of atomic markers as local probes for the dynamics of the guest-host interaction.

Benzoates↗
PubMed2004

Absence of intrinsic electric conductivity in single dsDNA molecules.

The intrinsic dc conductivity of long, individual lambda phage dsDNA molecules has been investigated by ultrasensitive low current-voltage-spectroscopy (IV) under ambient conditions and controlled low humidity inert gas atmosphere on microfabricated metal-insulator-metal gap structures. We found a strong dependence of the measured conductivity on the apparent humidity, which we attribute to capillary condensation of water to the immobilized DNA molecules, giving rise to additional ionic currents. Additional IV-spectroscopy experiments under controlled argon atmosphere always revealed a significant drop in electrical conductivity to 4 x 10(-15)AV(-1)microm(-1), indicating almost no considerable contribution of electrical long range charge transport.

Bacteriophage lambda↗
PubMed2001

c-erbB-2 expression in small-cell lung cancer is associated with poor prognosis.

Small-cell lung cancer (SCLC) carries a bad prognosis despite good initial response to chemotherapy. It is therefore important to identify molecular markers that influence survival as potential new therapeutic targets. In our study, expression of the tyrosine kinase c-erbB-2 (HER2/neu) receptor in tumor tissues of 107 consecutive newly diagnosed patients with primary SCLC was quantified using a monoclonal antibody directed against the c-terminal domain of c-erbB-2. A clear-cut positive expression of c-erbB-2 was observed in 13% of patients. Surprisingly, c-erbB-2 was an independent prognostic factor (RR = 2.16; p = 0.014) when a proportional-hazard model was adjusted to stage (limited vs. extensive disease) and performance status (WHO I-IV), the most relevant clinical parameters. Similarly, a significant association between c-erbB-2 and survival was obtained if a larger number of clinical parameters were included into the analysis, namely response to chemotherapy, TNM stage, lactate dehydrogenase (LDH), neuron-specific enolase (NSE), gender and age (p = 0.033). Interestingly, c-erbB-2 expression was more relevant for patients with advanced tumors. In the subgroup of patients with bad performance status (WHO II-IV), median survival of patients with undetectable c-erbB-2 expression was 274 days compared with only 23 days for patients with clear-cut positive c-erbB-2 immunohistochemistry (p = 0.0031; log-rank test). Similar results were obtained for patients with extensive disease (p = 0.028) and high TNM stages (T>2 or N>1 or M1; p < 0.068, all comparisons). In contrast, c-erbB-2 expression was not associated with survival in patients with limited disease (p = 0.97), low TNM stages (p > 0.56, all comparisons) and good performance status (p = 0.97). In conclusion, c-erbB-2 is expressed in more than 10% of SCLC. Expression of c-erbB-2 is an independent prognostic factor of survival. The effect of c-erbB-2 expression seems to become more important in advanced stages of the disease. Since c-erbB-2 is a therapeutical target in other types of cancer, further studies to identify the role of c-erbB-2 in SCLC are clearly warranted.

Age Factors↗
PubMed2001

Signal transduction pathways underlying the expression of tissue factor and thrombomodulin in promyelocytic cells induced to differentiate by retinoid acid and dibutyryl cAMP.

Acute promyelocytic leukaemia (APL) may be associated with disseminated intravascular coagulation, as a result of increased tissue factor (TF) expression and reduced thrombomodulin (TM) expression by APL blast cells. During retinoid acid (RA)- and dibutyryl cAMP (dbcAMP)-induced differentiation of the APL cells, there is a marked up-modulation of both the protein kinase A (PKA) and C (PKC) activities. In order to further assess whether these kinases are intimately associated with both the differentiation process and the regulation of TF and TM expression, we have correlated the modulation of their respective pathways with the extent of differentiation and modulation of these cellular receptors. NB4 cells were incubated with all-trans-RA (ATRA) or dbcAMP for up to 48 h. The contribution of phospholipase C (PLC), inositol phosphate (IP), PKC and PKA in the expression of CD11b, TF and TM was studied by the use of specific inhibitors. Myo-inositol uptake and PKC activity increased in cells induced to differentiate by ATRA but the retinoid did not affect cAMP levels or PKA activity. Under treatment with dbcAMP, PKA activity was increased while inositol uptake and PKC activity remained unchanged. Our results show that the effects of ATRA and dbcAMP on promyelocytic cells are closely related, respectively, to the PLC/IP/PKC and the cAMP/PKA pathways. In cells induced to differentiate by ATRA, CD11b expression seems more closely related to inositol uptake than to PKC activity while the expression of TF and TM show the opposite pattern, which suggests cellular events regulated at a different level within a common signal transduction pathway.

Bucladesine↗
PubMed2000

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates.

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Amino Acid Substitution↗
PubMed1999

Prevalence of bacterial vaginosis and correlation of clinical to Gram stain diagnostic criteria in low risk pregnant women.

The present study aimed to evaluate the prevalence of bacterial vaginosis, and the correlation of clinical Amsel criteria with Gram Nugent criteria for the diagnosis of bacterial vaginosis in a low risk population of pregnant women. Pregnant women under 28 weeks of gestation who were followed in the low risk clinics at two centers were evaluated for the presence of bacterial vaginosis using the Amsel clinical criteria, and underwent vaginal samples for Gram staining. Gram smears were examined for the diagnosis of bacterial vaginosis on the basis of Nugent's criteria. A total of 492 women were included in the study. Bacterial vaginosis was diagnosed in 1.6% (8/492) women on the basis of clinical criteria, and in 4.5% (22/492) according to Gram stain. The sensitivity and specificity of Amsel criteria compared with Gram stain were 35% and 99%, respectively. In accordance with other recent reports, the prevalence of bacterial vaginosis appears to be much lower in certain areas than figures previously suggested. In these populations, the correlation of composite clinical criteria defined in groups with high prevalence of bacterial vaginosis appears to be also poor.

Coloring Agents↗
PubMed1998

Antigen binding forces of individually addressed single-chain Fv antibody molecules.

Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.

Antigen-Antibody Reactions↗
PubMed1998

Altered Na+ and Li+ homeostasis in Saccharomyces cerevisiae cells expressing the bacterial cation antiporter NhaA.

The bacterial Na+ (Li+)/H+ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na+, Li+) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H+ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na+, Li+) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H+ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionally of plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase.

Bacterial Proteins↗
PubMed1998

Epidemiology and prevalence of seropositivity for hepatitis C virus in pregnant women in Granada.

OBJECTIVES: To study personal and familial antecedents of risk and prevalence of infection by HCV in pregnant women in the south area of Granada. PATIENTS AND METHODS: We included in the study 3003 pregnant women of the south area of Granada during the period from January 1993 to December 1995. Anti-HCV was detected in the third trimester of pregnancy by second and third generation ELISA, and positive results were confirmed by RIBA 3. We also determined HCV-RNA and genotype. Finally, we analyzed ALT levels in 1171 (39%) pregnant women. We carried out an epidemiological survey of all pregnant women, which included the following personal antecedents: transfusion, intravenous drug use, liver diseases, risk profession and sexually transmitted diseases. We studied the same antecedents in the parents, husbands and other relatives. RESULTS: Prevalence of anti-HCV was 0.63% (19 cases) with ELISA and 0.53% with RIBA. HCV-RNA was positive in 14 (74%) genotype 1b (57%) being the most frequent. ALT was increased in 52 (4.4%) pregnant women, 7 (13.5%) of whom were anti-HCV positive, versus 12 women (1%) in the normal ALT group (p < 0.001). In the epidemiological study we observed statistically significant differences in: a) housing characteristics [2125 (71%) anti-HCV negative pregnant women living in occupant-owned housing versus 7 (36%) in anti-HCV-positive group, p < 0.001]; b) personal antecedents of transfusion, chronic or acute hepatitis, or intravenous drug use (p < 0.001) (these factors were confirmed in the multivariable analysis), and c) familial antecedents of the husband (p < 0.05). CONCLUSIONS: In this study we demonstrated that 0.53% of the pregnant women were infected by HCV; most of them were HCV-RNA positive and was genotype 1b was the most frequent. The risk factors most frequently associated with infection were antecedents of transfusion, intravenous drug use and acute or chronic hepatitis.

Female↗
PubMed1996

Evidence for a selective and electroneutral K+/H(+)-exchange in Saccharomyces cerevisiae using plasma membrane vesicles.

The existence of a K+/H+ transport system in plasma membrane vesicles from Saccharomyces cerevisiae is demonstrated using fluorimetric monitoring of proton fluxes across vesicles (ACMA fluorescence quenching). Plasma membrane vesicles used for this study were obtained by a purification/reconstitution protocol based on differential and discontinuous sucrose gradient centrifugations followed by an octylglucoside dilution/gel filtration procedure. This method produces a high percentage of tightly-sealed inside-out plasma membrane vesicles. In these vesicles, the K+/H+ transport system, which is able to catalyse both K+ influx and efflux, is mainly driven by the K+ transmembrane gradient and can function even if the plasma membrane H(+)-ATPase is not active. Using the anionic oxonol VI and the cationic DISC2(5) probes, it was shown that a membrane potential is not created during K+ fluxes. Such a dye response argues for the presence of a K+/H+ exchange system in S. cerevisiae plasma membrane and established the non-electrogenic character of the transport. The maximal rate of exchange is obtained at pH 6.8. This reversible transport system presents a high selectivity for K+ among other monovalent cations and a higher affinity for the K+ influx into the vesicles (exit from cells). The possible role of this K+/H+ exchange system in regulation of internal potassium concentration in S. cerevisiae is discussed.

Biological Transport, Active↗
PubMed1995

The plant inorganic pyrophosphatase does not transport K+ in vacuole membrane vesicles multilabeled with fluorescent probes for H+, K+, and membrane potential.

It has been claimed that the inorganic pyrophosphatase (PPase) of the plant vacuolar membrane transports K+ in addition to H+ in intact vacuoles (Davies, J. M., Poole, R. J., Rea, P. A., and Sanders, D. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11701-11705). Since this was not confirmed using the purified and reconstituted PPase consisting of a 75-kDa polypeptide (Sato, M.H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., and Yoshida, M. (1994) J. Biol. Chem. 269, 6725-6728), these authors proposed that K+ transport by the PPase is dependent on its association with other membrane components lost during purification. We have examined the hypothesis of K+ translocation by the PPase using native vacuolar membrane vesicles from Vitis vinifera suspension cells, multilabeled with fluorescent probes for K+, H+, and membrane potential. This material contained a high proportion of right-side-out, tightly sealed vesicles, exhibiting high PPase activity which was strongly stimulated by uncouplers and K+. Proton pumping occurred in response to pyrophosphate addition in the absence of K+. No K+ incorporation into the vesicles could be observed after PPase energization in the presence of K+, although H+ transport was highly stimulated. The hydrolytic activity was stimulated by a protonophore and by a H+/K+ exchanger but not by the K+ ionophore valinomycin. No evidence could be obtained supporting the operation of an endogenous K+/H+ exchanger capable to dissipate the putative active K+ flux generated by the PPase. We conclude that PPase in native vacuolar membrane vesicles does not transport K+.

Enzyme Activation↗
PubMed1995

[Puerperal endometritis: study of 52 clinically and microbiologically diagnosed cases].

BACKGROUND: To know the epidemiologic features, clinical manifestations and etiology of puerperal endometritis in our environment in addition to the use of endometrial cultures in the microbiologic diagnosis of this infection. METHODS: A retrospective study of 52 cases of puerperal endometritis, clinically and microbiologically diagnosed over a 4-year period. RESULTS: Fifty percent of patients had undergone a cesarean, 36.5% presented puerperal anemia and 23% presented upper amniorrhexis at 12 hours. All the patients presented puerperal fever greater than 38 degrees C, 46% uterine subinvolution and 24% fetid lochia. The most frequently isolated microorganisms in the endometrial aspirate were Escherichia coli, Streptococcus agalactiae and Bacteroides spp. The association of ampicillin plus cefoxitine and clindamycin plus tobramycin presented in vitro efficacy in more than 85% of the isolates. CONCLUSIONS: The practice of endometrial aspirate and blood cultures in patients suspected of having puerperal endometritis effectively contributes to the diagnosis and treatment of this infection.

Adult↗
PubMed1993

[Perinatal Streptococcus agalactiae infections. Clinical epidemiological study and evaluation of a prevention program].

BACKGROUND: To prove both the importance of SGB (group B streptococci) as a cause of perinatal infection and the efficacy of a prophylactic treatment in pregnant women with cervicovaginal colonization by SGB. METHODS: Retrospective study of 197 third trimester pregnant women who were carriers of SGB (155 received intrapartum prophylaxis) and of 44 patients with SGB infections during pregnancy, post-partum and neonatal periods. RESULTS: No neonatal sepsis was detected in the group of SGB carrier mothers who received antibiotic prophylaxis. In carrier pregnant women who did not receive prophylaxis, one case of neonatal sepsis by SGB was detected and a greater prevalence of intrapartum fever, and neonatal infection with negative cultures was observed. SGB was frequently isolated as a cause of early sepsis and neonatal meningitis (13 cases), intraamniotic infection (12 cases) and puerperal endometritis (8 cases). In 45% of the patients with perinatal infections by SGB, the cervicovaginal culture performed in the third trimester of pregnancy did not detect the presence of SGB. CONCLUSIONS: The administration of intrapartum ampicillin to pregnant SGB carriers permits the prevention of perinatal infections by this microorganism in a great number of patients, although the possibility of late colonization, which may not be detected during pregnancy, stil remains.

Ampicillin↗
PubMed1991

[Haemophilus influenzae and genital infection].

We report nine cases of H. influenzae genital infections in women. Six patients had a total of 7 episodes of Bartholin glands abscess. One patient developed a post-caesarean endomyometritis on the 16th post-delivery day. The remaining patient developed an amniotic fluid infection in the 32nd week of pregnancy. All the nine H. influenzae strains were sensitive to ampicillin. We assess the rising importance of H. influenzae as an etiologic agent of female genital tract infections.

Abscess↗