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Biomedical subjects

M Ramos

Publications and source records attributed to M Ramos.

At least 37 records · Page 2Linked to original sources
PubMed2004

A genistein-sensitive Na+/Ca2+ exchange is responsible for the resting [Ca2+]i and most of the Ca2+ plasma membrane fluxes in stimulated rat cerebellar type 1 astrocytes.

The differential role of Na+/Ca2+ exchange in the regulation of intracellular ionized calcium ([Ca2+]i) in immunological and pharmacologically identified type 1 astrocytes and Purkinje cells was studied in rat cerebellar culture, using Ca2+ (Fluo-3, Fura-2) and Na+ (SBFI) fluorescence measurements. The mean resting [Ca2+]i was significantly higher (191 +/- 8 nM, n=25) in type 1 astrocytes than in Purkinje cells (92 +/- 2.5 nM, n=35). In contrast to Purkinje cells, in unstimulated cerebellar type 1 astrocytes, forward and reverse Na+/Ca2+ modes operate under resting physiological conditions, being responsible for most of the total Ca2+ transplasma membrane fluxes. Four observations support this hypothesis: (1) under resting conditions of temperature and ionic composition, Na+o removal causes a remarkable increase in [Ca2+]i, being inhibited by 2',4' dichlorobenzamil (DCB), and 2-[2-[4-(nitrobenzilloxiphenyl ethyl] isothiourea metanesulfonate (KB-R7943); (2) Ca2+o removal in the presence of Na+o causes an important drop in [Ca2+]i, which is absent in Li+o or NMG+o (N-methyl-D-glucamine) containing medium; (3) the reverse mode exchange inhibitor KB-R7943 mimics the removal of Ca2+o only in the presence of Na+o; and (4) under loaded [Na+]i conditions (ouabain or the activation of taurine-Na+-cotransport), reverse mode exchange increases in both astrocytes and Purkinje cells. In type 1 astrocytes stimulated with endothelin-3 (ET-3), the recovery of the Ca2+i signal occurs largely through the Na+/Ca2+ exchanger. Genistein, a tyrosine kinase inhibitor, completely and reversibly blocks all exchange activity, but not its inactive analogue daidzein, thus suggesting that the Na+/Ca2+ exchanger of cerebellar type 1 astrocytes may be modulated by phosphorylation. Our main conclusion is that in rat cerebellar type 1 astrocytes under resting physiological conditions, most of the total transplasma membrane Ca2+ fluxes take place through the Na+/Ca2+ exchanger, thus accounting for the resting [Ca(2+)]i.

Animals↗
PubMed2004

Detection of milk mixtures in Halloumi cheese.

A capillary electrophoresis method has been applied to the detection of illegal addition of milk from goat and/ or cow in Halloumi cheese, traditionally made with sheep milk. The electrophoretic profiles of the casein from Halloumi cheeses have revealed that caprine para-kappa-casein and bovine alphas1-casein peaks point to the presence of low percentages of goat's and/or cow's milk added to Halloumi cheese. Stepwise multiple linear regression has been used to predict these percentages with a standard error of the estimation of 2.14%. The analytical method combined with the statistical application is valid for the prediction of percentages higher than 2% of goat's and percentages of 5% of cow's milk added to the cheese either in fresh or ripened cheese. The standard error of estimation was higher for the prediction of cow's milk than for goat's milk.

Animals↗
PubMed2004

Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis.

The hydrolysis of crude egg white with pepsin, trypsin, and chymotrypsin produced peptides with angiotensin-converting enzyme (ACE) inhibitory properties. These peptides were mainly derived from the proteolysis of ovalbumin. The most active hydrolysates were obtained after treatment with pepsin (50% inhibitory concentration [IC50], 55.3 microg/ml), with the fraction having a molecular mass lower than 3,000 Da giving the highest ACE inhibitory activity (IC50, 34.5 microg/ml). Nine subfractions were collected from the fraction with a molecular mass lower than 3,000 Da using semipreparative reversed-phase high-performance liquid chromatography. Considerable ACE inhibitory activity (IC50 < 40 microg/ml) was found in three of them. These subfractions were analyzed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry, and 14 peptides were identified. These sequences were synthesized, and their ACE inhibitory activities were measured. Among the identified peptides, two novel sequences with potent ACE inhibitory activity were found. The amino acid sequences of these inhibitors were identified as Arg-Ala-Asp-His-Pro-Phe-Leu and Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu and showed IC50 values of 6.2 and 4.7 microM, respectively.

Amino Acid Sequence↗
PubMed2003

Mass mapping analysis as a tool for the identification of genetic variants of bovine beta-casein.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometric analysis of the tryptic digest of beta-casein A2 and beta-casein B was performed before and after the separation of the peptides by LC. The overlapping of the chromatograms showed that all peaks were present in both samples, except for one only found in the tryptic digest of the A2 variant and two in the B variant. Experimental masses could be assigned to those peptides produced by tryptic digest of beta-casein variant. This peptide mapping strategy and current methodological improvements represent a promising tool for the identification of milk genetic variants with the difference of an amino acid substitution.

Amino Acid Sequence↗
PubMed2003

Determination of chloramphenicol residues in shrimps by liquid chromatography-mass spectrometry.

A liquid chromatographic method with mass spectrometric detection and identification (LC-MS) is presented for the determination of chloramphenicol (CAP) in shrimp tissues. Homogenized shrimp samples were extracted with phosphate buffer (pH 7.0). Clean-up was carried out on a C(18) SPE cartridge. Chloramphenicol was determined by LC-MS-ESI in negative mode. The column used was a Symmetry Shield with a mixture of acetonitrile-water (25:75) as mobile phase. Shrimp samples were fortified at CAP levels between 0.2 and 50 ng g(-1) with 5D-CAP as internal standard. At these levels, accuracies lay between 101 and 110% and between-day reproducibilities were lower than 7.1%, expressed as the variation coefficient of the mean. Limit of decision (CCalpha) was 0.02 ng g(-1). Limit of quantitation (LOQ) was 0.2 ng g(-1).

Animals↗
PubMed2003

Weekly docetaxel as second-line therapy for patients with advanced breast cancer resistant to previous anthracycline treatment.

This phase II trial evaluated the efficacy and toxicity of weekly docetaxel as treatment of advanced metastatic breast cancer patients resistant to prior anthracycline chemotherapy. After the first 18 patients, the initial dose (40 mg/m2, 30-min i.v. infusion for 6 consecutive weeks, followed by 2-week rest) was reduced to 36 mg/m2 in the remaining 17 patients due to the incidence of toxicity (28% grade 3-4 asthenia). Overall response rate was 34% (95% CI, 19-50): two complete (6%) and ten partial responses (28%) were found. The median duration of response was 6.8 months, the median time to disease progression was 8.4 months, and the median overall survival was 13.6 months (median follow-up of 11.4 months). Neutropenia was the only severe hematologic toxicity (17% of patients), whereas asthenia, nail, ocular and skin disorders were the most common nonhematologic toxicities. Only one death during further follow-up was related to toxicity (caused by pulmonary fibrosis). In conclusion, we found weekly docetaxel to be an active and safe chemotherapy regimen for patients with metastatic breast resistant to previous anthracyclines. This weekly regimen caused minimal myelosupression, while retaining significant activity against advanced breast cancer. Both factors provide attractive possibilities for the development of combination therapies incorporating weekly docetaxel. Nevertheless, the number of patients receiving either dose (40 and 36 mg/m2) which we studied is low and our results require confirmation on larger groups of patients.

Adult↗
PubMed2003

Influence of proteolysis of milk on the whey protein to total protein ratio as determined by capillary electrophoresis.

Capillary electrophoresis (CE) was used to determine the whey protein to total protein ratio in raw and UHT milk samples with different degrees of proteolysis caused by storage. In raw milks, the analysis of samples taken at regular times demonstrated the influence of proteolysis in the whey protein to total protein determination, which was overestimated after 4 d of storage. In UHT milks, the overestimation of the whey protein to total protein ratio took place after 30 or 60 d of storage. However, the ratios alphaS1-CN/beta-CN and alphas1-CN/kappa-CN permitted detection of the samples of raw or UHT milk with degraded proteins. The distorted capillary electrophoretic pattern obtained for UHT milks made necessary an integration of the electropherograms in a "valley-to-valley" way. Results for raw milk samples were identical when "valley-to-valley" was compared to standard integration techniques. This CE method could be considered an alternative method to derivative spectroscopy for the determination of the whey protein to total protein of milk and could be used to detect samples with proteolysis.

Animals↗
PubMed2002

HLA-B27 and the pathogenesis of spondyloarthritis.

The association of HLA-B27 with ankylosing spondylitis and other spondyloarthropathies ranks among the strongest between any HLA antigen and a human disease. Yet, in spite of intense research and advanced knowledge of the biochemistry and biology of major histocompatibility complex molecules, the mechanism of this association remains unknown. This review attempts a critical assessment of current pathogenetic hypotheses from evidence concerning the epidemiology of HLA-B27 association with disease, its peptide-binding specificity, and other aspects of the molecular biology and immunology of this molecule.

HLA-B27 Antigen↗
PubMed2002

[Isotopic renal study in diabetic nephropathy].

UNLABELLED: This study aimed to evaluate the prognostic value of the kidney split function (KSF), the effective renal plasma flow (ERPF) and glomerular filtration rate (GF) in the evolution of diabetic nephropathy. MATERIAL AND METHODS: 125 patients (IDDM:10, NIDDM:115), including 62 men, ages 18-84 years, were studied. In all cases GF (Cr 51 EDTA), ERPF (131I-hippuran), kidney split function (obtained from the 99mTc-MAG3 renogram) and basal serum creatinine (SC) were evaluated at the beginning of the study and patients were clinically followed up for 5 years by SC. Ultrasound and/or intravenous digital angiography were performed in 120 patients. RESULTS: In patients with decreased ERPF (76 patients) the SC increased progresively during the 5 years, in both those with elevated PC (N = 54) as well as in those with normal baseline SC (n = 22). In patients with normal ERPF the SC did not change significantly during the study. An asymethric split function (KSF < 40% in one kidney) was found in the renogram of 42 patients; in 32 of them the ERPF was decreased. Of these, ulttrasonography was normal in 9, and vascular and/or obstructive pathology was demonstrated by ultrasonography or digital angiography in only 6 patients. CONCLUSION: The ERPF becomes altered earlier than serum creatinine in patients with diabetic nephropaty. ERPF and split function calculation, and FG seems to have higher prognostic value than ultrasounds in the management of diabetic nephropathy. Radionuclides seems to be an accurate test than the ultrasonography to detect renal function abnormalities in patients with diabetic and thus can be an examination of choice in these patients.

Adolescent↗
PubMed2001

Identification of novel HLA-B27 ligands derived from polymorphic regions of its own or other class I molecules based on direct generation by 20 S proteasome.

HLA-B27 is strongly associated with ankylosing spondylitis. Natural HLA-B27 ligands derived from polymorphic regions of its own or other class I HLA molecules might be involved in autoimmunity or provide diversity among HLA-B27-bound peptide repertoires from individuals. In particular, an 11-mer spanning HLA-B27 residues 169-179 is a natural HLA-B27 ligand with homology to proteins from Gram-negative bacteria. Proteasomal digestion of synthetic substrates demonstrated direct generation of the B27-(169-179) ligand. Cleavage after residue 181 generated a B27-(169-181) 13-mer that was subsequently found as a natural ligand of B*2705 and B*2704. Its binding to HLA-B27 subtypes in vivo correlated better than B27-(169-179) with association to spondyloarthropathy. Proteasomal cleavage generated also a peptide spanning B*2705 residues 150-158. This region is polymorphic among HLA-B27 subtypes and class I HLA antigens. The peptide was a natural B*2704 ligand. Since this subtype differs from B*2705 at residue 152, it was concluded that the ligand arose from HLA-B*3503, synthesized in the cells used as a source for B*2704-bound peptides. Thus, polymorphic HLA-B27 ligands derived from HLA-B27 or other class I molecules are directly produced by the 20 S proteasome in vitro, and this can be used for identification of such ligands in the constitutive HLA-B27-bound peptide pool.

Amino Acid Sequence↗
PubMed2001

Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis.

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.

Animals↗
PubMed2001

Capillary electrophoresis for the analysis of food proteins of animal origin.

In recent years, capillary electrophoresis (CE) has become an analytical technique with many applications in the study of food proteins and peptides. This review describes the existing CE methods of analysis of milk, egg, meat and fish proteins and peptides. The major developments in the application of CE to solve different problems in food technology, such as the assessment of technological processes, quality, and authenticity control of animal foods, are considered. A section dealing with future directions on the analysis of food proteins by CE is also included.

Animals↗
PubMed2001

Minimal alterations in the HLA-B27-bound peptide repertoire induced upon infection of lymphoid cells with Salmonella typhimurium.

OBJECTIVE: To characterize putative changes in the HLA-B27-bound peptide repertoire following infection of lymphoid cells with Salmonella typhimurium, a bacterium known to trigger reactive arthritis in HLA-B27-positive individuals. METHODS: A protocol was developed for efficient large-scale infection of lymphoblastoid cell transfectants expressing HLA-B*2705. HLA-B27-bound peptide pools were isolated from noninfected and infected B*2705+ cells and comparatively analyzed by high-performance liquid chromatography. Peptide-containing chromatographic fractions from noninfected and infected cells were systematically compared by mass spectrometry (MS) to look for putative differences at the level of individual peptides. RESULTS: The presence of B*2705 did not influence S typhimurium invasion, since this was equally efficient in nontransfected or B27-transfected cells. The chromatographic profiles of B*2705-bound peptides from noninfected and infected cells were virtually identical. A total of 808 molecular species were compared by MS. Of these, 807 were present in both infected and noninfected cells. Only one molecular species from infected cells lacked a detectable counterpart in noninfected cells. CONCLUSION: Intracellular infection of lymphoid cells by S typhimurium induces minimal alterations in the HLA-B27-bound peptide repertoire. Minor changes detectable by cytotoxic T lymphocytes, but not easily amenable to direct biochemical analysis, are not ruled out.

Arthritis, Reactive↗
PubMed2001

Trypanosoma evansi: a convenient model for studying intracellular Ca(2+) homeostasis using fluorometric ratio imaging from single parasites.

The aim of this work was to measure, for the first time, the basal cytosolic Ca(2+) levels of Trypanosoma evansi and to explore the possibility of observing changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) using fluorescence ratio imaging techniques in single isolated parasites of this species. Under appropriate loading conditions, the high intracellular levels of the Ca(2+) fluorescence probe Fura-2 permits resolution, in real time, of single parasite [Ca(2+)](i) signals. Measurements of the basal [Ca(2+)](i) indicate that homeostatic mechanisms maintain [Ca(2+)](i) at 106 +/- 38 (n = 32) nM in the presence of 2 mM extracellular calcium. The resting [Ca(2+)](i) was unaffected by changes in extracellular Ca(2+) in the range from 0 to 10 mM. The Ca(2+) ionophore A23187 induced a large increase in [Ca(2+)](i) which (i) reached a steady state value even in the simultaneous presence of both external calcium and ionophore and (ii) returned to base line upon removal of extracellular Ca(2+). A dose-response curve of the protonophore nigericin shows that T. evansi contains an important pH-sensitive intracellular pool which may be released by this drug with a K(1/2) of 8 microM. These data demonstrate that this parasite contains highly efficient systems to control [Ca(2+)](i). Finally, our results, with the use of sera as source of an antibody-complement to induce Ca(2+) entry, demonstrate that it is possible to resolve fast [Ca(2+)](i) signals in single parasites from T. evansi.

Animals↗
PubMed2001

Oxidative stress induces the expression of the major histocompatibility complex in murine tumor cells.

The effect of t-butyl hydroperoxide (t-BOOH) on the induction of the Major Histocompatibility Complex (MHC) class I genes has been studied in two cell clones (B9 and G2) of the methylcholanthrene-induced murine fibrosarcoma GR9. These two clones were selected based on their different biological and biochemical behavior specially related to their tumor induction capability when injected into a BALB/c mouse. t-BOOH (0.125 mM) induced the expression of H-2 molecules in both cell clones. In B9 cell clone, in which MHC basal expression is very low or absent, t-BOOH significantly induced H-2Kd, H-2Dd and H-2Ld molecules. In G2 cell clone the expression of MHC class I genes was also enhanced by the xenobiotic, the effect being especially significant on the H-2Ld molecule which is not expressed under basal conditions. H-2 molecules expression was accompanied by the activation of the transactivator factor NF kappa B. These results suggest that oxidative stress may modulate the antigen expression of tumor cells and thus the immune response of the host organism. Basal levels of oxidative parameters, such as anti-oxidant enzymes, malondialdehyde (MDA) and the DNA damaged base 8-hydroxy-2'-deoxyguanosine (8-OHdG), showed differences between the two fibrosarcoma cell clones.

3T3 Cells↗
PubMed2001

Phase I clinical evaluation of a neutralizing monoclonal antibody against epidermal growth factor receptor in advanced brain tumor patients: preliminary study.

High levels of growth factors and their receptors have been demonstrated in human tumors. Gliomas and meningiomas are characterized by overexpression of epidermal growth factor receptor (EGF-R). Ior egf/r3, is a neutralizing murine monoclonal antibody (MAb) against EGF-R, and was generated at the Cuban Institute of Oncology. The antibody recognizes EGF-R with high affinity, inhibiting tyrosine kinase activation. A clinical trial was conducted in brain tumor patients to evaluate toxicity, immunogenicity, and clinical benefit of escalating doses of the antibody. Nine patients with histologically confirmed gliomas or meningiomas, who had active or recurrent disease after receiving conventional treatment, received four intravenous doses of ior egf/r3. Total dosages ranged from 160 to 480 mg. As inclusion criteria, radioimmunoscintigraphy with the same MAb labeled with 99mTechnetium (99mTc) was performed. Immune response against the murine antibody was also evaluated. After four doses of ior egf/r3 MAb, no significant toxicity was found, except in one patient who developed a grade 4 allergic adverse event. This reaction was probably related with previous sensitization to the same MAb and the development of human anti-mouse antibodies (HAMA) response. Despite no major objective antitumor responses, eight patients had stable disease on the 6-month evaluation, and two patients remain alive after four years of MAb therapy.

Adult↗
PubMed2001

Phase I clinical evaluation of a neutralizing monoclonal antibody against epidermal growth factor receptor.

Ior egf/r3, a neutralizing monoclonal antibody (mAb) against Epidermal Growth Factor Receptor (EGFR) was generated at the Cuban Institute of Oncology. Immunoscintigraphic studies in 148 patients with this 99-m Technetium (99Tc) labeled mAb, showed a high sensitivity and specificity for in vivo detection of epithelial tumors. To study safety, pharmacokinetic and immunogenicity of ior egf/r3 at high doses, a phase I clinical trial was conducted. Nineteen patients with advanced epithelial tumors received 4 mAb intravenous infusions at 6 dose levels: from 50 to 500 mg. Previously, immunoscintigraphic images using the same mAb labeled with 99Tc were acquired. Blood samples were collected for pharmacokinetic analysis and HAMA response. After mAb therapy, objective response was classified according to WHO criteria. Ior egf/r3 was well tolerated in spite of the high-administered doses. Only a severe adverse reaction consisting of hypotension and lethargy was observed. In 13 patients, selective accumulation of 99Tc-labeled mAb was observed at the site of the primary tumor or the metastasis. Pharmacokinetic analysis revealed that elimination half-life and the area under the time-concentration curve increased linearly with dose. HAMA response was detected in 17 patients. After 6 months of mAb therapy, 4 patients had stable disease. One patient had a tumor partial remission after 3 cycles of ior egf/r3.

Adult↗