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Biomedical subjects

A Castro

Publications and source records attributed to A Castro.

At least 127 records · Page 7Linked to original sources
PubMed1998

Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase.

Phosphorylation of the RII regulatory subunits of cyclic AMP-dependent protein kinases (PKAs) was examined during the HeLa cell cycle. Three RIIalpha isoforms of 51, 54, and 57 kDa were identified by RIIalpha immunodetection and labeling with 8-azido[32P]cAMP in different cell cycle phases. These isoforms were characterized as different phosphorylation states by the use of selective PKA and cyclin-directed kinase inhibitors. Whereas RIIalpha autophosphorylation by PKA caused RIIalpha to shift from 51 to 54 kDa, phosphorylation of RIIalpha by one other or a combination of several kinases activated during mitosis caused RIIalpha to shift from 51 to 57 kDa. In vivo incorporation of [32P]orthophosphate into mitotic cells and RIIalpha immunoprecipitation demonstrated that RIIalpha was hyperphosphorylated on a different site than the one phosphorylated by PKA. Deletion and mutation analysis demonstrated that the cyclin B-p34(cdc2) kinase (CDK1) phosphorylated human recombinant RIIalpha in vitro on Thr54. Whereas RIIalpha was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RIIalpha from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RIIalpha, and this may alter its subcellular localization.

Affinity Labels↗
PubMed1998

Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts.

The Xenopus homologue of Drosophila Fizzy and budding yeast CDC20 has been characterized. The encoded protein (X-FZY) is a component of a high molecular weight complex distinct from the APC/cyclosome. Antibodies directed against FZY were produced and shown to prevent calmodulin-dependent protein kinase II (CaMKII) from inducing the metaphase to anaphase transition of spindles assembled in vitro in Xenopus egg extracts, and this was associated with suppression of the degradation of mitotic cyclins. The same antibodies suppressed M phase-promoting factor (MPF)-dependent activation of the APC/cyclosome in interphase egg extracts, although they did not appear to alter the pattern or extent of MPF-dependent phosphorylation of APC/cyclosome subunits. As these phosphorylations are thought to be essential for APC/cyclosome activation in eggs and early embryos, we conclude that at least two events are required for MPF to activate the APC/cyclosome, allowing both chromatid segregation and full degradation of mitotic cyclins. The first one, which does not require FZY function, is the phosphorylation of APC/cyclosome subunits. The second one, that requires FZY function (even in the absence of MAD2 protein and when the spindle assembly checkpoint is not activated) is not yet understood at its molecular level.

Amino Acid Sequence↗
PubMed1998

Administration to mouse of endotoxin from gram-negative bacteria leads to activation and apoptosis of T lymphocytes.

Lipopolysaccharide (LPS) from gramnegative bacteria is a well-known T cell-independent B lymphocyte mitogen and macrophage/monocyte activator. While the conventional view holds that LPS is ignored by T cells, we report here that administration of LPS to mice activates all B cells, but also engages most CD4 and CD8 T cells, as measured by the expression of the activation markers CD69 and CD25 and by size increase. T cells recruited in endotoxin-treated mice showed, following in vitro stimulation by concanavalin A, altered patterns of cytokine production. In vivo, massive T cell apoptosis was evidenced in the days following LPS exposure. The present observation may contribute novel insights into the mechanisms of endotoxin shock and of the immunological consequences of gram-negative infections.

Animals↗
PubMed1998

Use of a microquantity enzyme immunoassay in a large-scale study of measles, mumps and rubella immunity in Italy.

The seroprevalence of antibodies to measles, mumps, and rubella viruses (MMR) was determined in 1498 subjects in Catania, Italy, ranging in age from 1 month to 25 years. The study population was divided into seven age groups and screened by enzyme immunoassay using microquantities (10 microl) of whole blood collected by fingerprick on filter paper discs. The results showed that seroconversion for measles (87.6%) and mumps (73.2%) occurred between 6 and 10 years of age. The seroprevalence of antibodies to rubella virus increased slowly through the age groups, reaching the highest rate (93.3%) between 16 and 20 years of age. Passively transmitted maternal antibodies to mumps and rubella were absent in babies between 5 and 8 months of age, and a few cases positive for measles antibodies were found among babies 6 and 7 months of age. The enzyme immunoassay was demonstrated to be suitable for low-cost large-scale screening for MMR immunity. The rate of vaccine failure was also evaluated and found to be 9.5% for the measles virus, 12.9% for the mumps virus and 0.0% for the rubella virus.

Adolescent↗
PubMed1998

Presence of angiotensin II and expression of angiotensin II type-2 receptor in human fallopian tube.

OBJECTIVE: To determine the presence of angiotensin II and angiotensin II type-2 receptor subtype messenger RNA (mRNA) in human fallopian tube. DESIGN: Frozen fallopian tubes were used for all studies. SETTING: Procedures were performed at the Institute of Maternal and Child Research, School of Medicine, University of Chile and San Borja-Arriarán Clinical Hospital, National Health Service, Santiago, Chile. PATIENT(S): Eight patients (30-38 years) undergoing surgical sterilization or hysterectomy for benign gynecologic conditions. INTERVENTION(S): Surgery was scheduled in the proliferative or secretory stage of the menstrual cycle. MAIN OUTCOME MEASURE(S): Localization of angiotensin II by immunofluorescence studies, expression of angiotensin II type-2 receptor mRNA, and concentrations of angiotensin II receptor subtypes and binding characteristics. RESULT(S): Angiotensin II was immunolocalized mainly in blood vessel endothelium and, to a lesser extent, in stromal cells. Both binding and angiotensin II type-2 receptor mRNA were detected at high levels. No differences were seen in receptor concentration, dissociation constants or median inhibitory concentration in fallopian tubes ipsilateral or contralateral to the corpus luteum. CONCLUSION(S): Angiotensin II and angiotensin II receptor are present in human fallopian tube. Angiotensin II type-2 receptor was the main receptor subtype. These results suggest that the renin-angiotensin system may play a regulatory role in functions of the fallopian tube.

Adult↗
PubMed1998

Luteal leukocytes are modulators of the steroidogenic process of human mid-luteal cells.

Flow cytometry analysis of luteal cells revealed that an important proportion of these cells are leukocytes. The percentage of leukocytes was higher in the early (42 +/- 4) and late (35 +/- 3) luteal phases than in the mid-luteal (24 +/- 2) phase. However, the proportion of macrophages did not differ between the luteal stages. The flow cytometric properties correlated with cellular size and granularity were not reliable as discriminators of luteal cell subpopulations. Therefore, to assess the contribution of luteal leukocytes, these cells were completely removed from luteal cell suspensions (total cells), by a negative selection procedure (immunomagnetic separation). The functional role of leukocytes in mid-luteal steroidogenesis was assessed, in total as well as leukocyte-depleted cells. Progesterone production was found to have increased 2.2-fold in leukocyte-depleted cell cultures, in comparison with total cells under basal conditions. However, the response to human chorionic gonadotrophin (HCG) was 36% lower under the latter conditions. Oestradiol production was not significantly modified under basal or HCG-treated conditions. In leukocyte-depleted cells, the concentration of interleukin (IL)-1beta decreased 5-fold in comparison with total cell cultures, suggesting that leukocytes are the principal source of IL-1beta. In summary, the results of the present investigation suggest functional interactions between the immune system and steroidogenic cells of the human corpus luteum.

Adult↗
PubMed1998

Low energy intracardiac cardioversion of persistent atrial fibrillation.

The aims of the study were to verify the efficacy and safety of low energy internal cardioversion (LEIC) in patients with persistent atrial fibrillation (AF) and to identify the factors affecting the atrial defibrillation threshold (ADT). Forty-nine patients with persistent (lasting > or = 10 days) AF underwent LEIC. In each patient, two 6 Fr custom-made catheters with large active surface areas were positioned in the coronary sinus (cathode) and the lateral right wall (anode), respectively, for shock delivery, and a tetrapolar lead was placed in the right ventricular apex for R wave synchronization. Truncated, biphasic (3 ms + 3 ms), exponential shocks were used, beginning at 50 V and increasing in steps of 50 V until sinus rhythm had been restored. Mild sedation (diazepam 5 mg i.v.) was administered to 12 patients. Sinus rhythm was restored in all the subjects with mean voltage and energy levels of 352.0 +/- 80.3 V and 8.2 +/- 3.4 J, respectively. The ADT in patients pretreated with amiodarone (6.4 +/- 1.8 J) was lower than that of patients who had not received any antiarrhythmic drugs (9.2 +/- 3.7) (P = 0.04). No ventricular arrhythmias were induced by any of the atrial shocks, and no other complications were observed. During a mean follow-up of 162.9 +/- 58.7 days, AF recurred in 21 (43%) patients; 71% of these occurred in the first week after cardioversion. LEIC is effective in restoring sinus rhythm in patients with persistent AF. The technique seems to be safe and does not require general anesthesia or, in most cases, sedation. Patients pretreated with amiodarone have lower ADTs.

Adult↗
PubMed1998

Treatment with the competitive NMDA antagonist GPI 3000 does not improve outcome after cardiac arrest in dogs.

BACKGROUND AND PURPOSE: We previously showed that treatment with a competitive N-methyl-D-aspartate (NMDA) receptor antagonist GPI-3000 (GPI) improved short-term physiological recovery after incomplete global cerebral ischemia complicated by dense acidosis. We tested the hypothesis that GPI administered after resuscitation from cardiac arrest would improve a more long-term recovery as measured by neurobehavioral assessment and neuropathology 4 days after resuscitation. METHODS: Anesthetized dogs were subjected to 7 minutes of cardiac arrest followed by vest cardiopulmonary resuscitation. Neurobehavioral outcomes were scored daily on a score ranging from 0 (normal) to 500 (worst). On the fourth day, the animals were killed, and neuropathology was evaluated in a blinded manner in the hippocampus and the neocortex by hematoxylin and eosin staining and by determination of percentage of injured neurons. Three groups of animals were treated in a randomized, blinded protocol with either saline (SAL), low-dose GPI (5 mg/kg followed by 1 mg/kg per hour for 2 hours), or high-dose GPI (25 mg/kg, followed by 5 mg/kg per hour for 2 hours). RESULTS: The mortality rate was higher in animals receiving GPI than in saline-treated control animals (4 of 15 deaths in SAL, 6 of 15 in the low-dose GPI group, and 9 of 18 in the high-dose GPI group). Neurobehavioral scores were depressed in GPI-treated animals compared with saline-treated control animals in a dose-dependent manner, with 96-hour scores of essentially normal (9+/-2) in saline-treated animals compared with those animals with significant impairment (181+/-47) treated with high-dose GPI. Neuropathological damage in the neocortex was most severe in GPI-treated animals, with the percentage of injured neurons dependent on the dose: 8.3%+/-2.7% SAL, 13.2%+/-6.4% low-dose GPI, and 39.4%+/-10.1%, high-dose GPI. CA1 neuronal damage was severe regardless of treatment. CONCLUSIONS: Contrary to results seen in experimental global and focal cerebral ischemia, in which NMDA receptor antagonism may improve responses to injury, receptor antagonism with GPI does not improve brain outcome after cardiac arrest and resuscitation in the dog. Behavioral and histological outcomes both were worsened by GPI treatment at two doses, and mortality was higher relative to saline control treatment. We speculate that systemic drug effects, as well as potential neurotoxicity of the drug under ischemic conditions, may be responsible for the deleterious outcomes observed in our cardiac arrest model.

Amino Acids↗
PubMed1998

Genetic characterization of APOB and D17S5 AFLP loci in a sample from the Basque Country (northern Spain).

VNTR polymorphism of AFLP loci D17S5 and APOB was analyzed in 100 unrelated individuals residing in the Basque Country by PCR amplification and polyacrylamide gel electrophoresis. Population parameters were estimated to validate these loci as routine markers in human identification. Thus, although both loci showed extensive polymorphism (with gene diversities of approximately 80% for both systems) and although concordance with Hardy-Weinberg equilibrium and linkage equilibrium was observed, comparisons with other populations displayed significant differences. Moreover, further analysis of neutral parameters limits this validation to D17S5 because APOB displays excesses of both the total number of alleles and the number of rare alleles; possible reasons for this discrepancy (presence of deleterious alleles, bottleneck effect, admixture, complexity of APOB polymorphism) are discussed.

Alleles↗
PubMed1998

[The efficacy of high- and low-energy intracavitary cardioversion in chronic atrial fibrillation refractory to transthoracic cardioversion].

BACKGROUND: The purpose of our study was to evaluate the efficacy and safety of intracavitary cardioversion (IC) with either high (HEIC) or low (LEIC) energy in patients with chronic atrial fibrillation (AF) refractory to external cardioversion. METHODS: Forty patients underwent IC with either high (30 pts) or low energy (10 pts); HEIC was performed by delivering one or more shocks between one electrode of the lead positioned in the right atrium and a back plate. The following energy values were used in sequence: 100, 200, 300 joules. In LEIC shocks were delivered between two custom-made decapolar leads (one in the coronary sinus and another one in the right lateral atrial wall) beginning from 50 Volts and increasing the voltage by steps of 50 Volts. Another catheter in the right ventricular apex was used for ventricular synchronization. RESULTS: Sinus rhythm was restored in 24/30 (80%) patients undergoing HEIC and in 10/10 (100%) patients undergoing LEIC. No serious or permanent complications occurred. CONCLUSIONS: HEIC and LEIC are effective in patients with AF refractory to external cardioversion. Both the procedures seem to be safe.

Adult↗
PubMed1998

Population genetics and forensic applications using multiplex PCR (CSF1PO, TPOX, and TH01) loci in the Basque Country.

A population study in a sample of 200 unrelated individuals from the Basque Country (Northern Spain) was carried out using the GenePrint STR Multiplex System. The PCR products were electrophorized on a denaturing polyacrylamide gel and visualized by silver staining. The loci are TH01, TPOX, and CSF1PO. All loci meet Hardy-Weinberg expectations, and independence of alelles at these STR loci was found. A comparison with other population groups appeared to indicate that frequencies are well conserved in Caucasians, but differ from those of other racial groups. We have also calculated Fst as a measure of population subdivision. No appreciable genetic subdivision in the Caucasian populations studied here was found. Some statistical parameters of forensic interest (Pex, PM and PD) were also calculated. No exclusions were found in 100 mother-child and father-child meiosis. To evaluate the applicability of these systems to forensic casework, we studied the minimum quantity of DNA which can be used applying the multiplex methodology, and the minimum quantity that can be typed in a mixed sample. We also examined several samples such as hair roots, semen stains, vaginal swabs, blood stains and temporary teeth, each of these of varying ages.

Alleles↗
PubMed1998

[Cardiorespiratory failure and diabetic autonomic neuropathy: a case report].

Advanced autonomic neuropathy in diabetic patients is associated with homeostatic cardiovascular response. Sympathetic compensation diminishes and the organism becomes more susceptible to the depressant effect of certain drugs. The incidence of undesirable cardiovascular effects (bradycardia, hypotension and sudden heart failure) during anesthesia is higher in such patients. We report the case of a woman with diabetes who suffered two episodes of sudden heart failure after brachial plexus block through the axilla, followed one year later by sudden heart failure during general anesthesia for kidney transplantation. The possible contributing mechanisms are described.

Adult↗
PubMed1997

Single-molecule detection of specific nucleic acid sequences in unamplified genomic DNA.

A new technique is described for the rapid detection of specific nucleic acid sequences in unamplified DNA samples. The method consists of using two nucleic acid probes complementary to different sites on a target DNA sequence. The two probes are each labeled with different fluorescent dyes. When mixed with a sample containing the target DNA, the two probes hybridize to their respective binding sites on the same target DNA molecule. The sample is then analyzed by a laser-based ultrasensitive fluorescence system capable of detecting single fluorescent molecules at two different wavelength channels simultaneously. Since the probes are bound to the same target DNA molecule, their signals appear simultaneously. Thus, coincident detection of both dyes provides the necessary specificity to detect an unamplified, single-copy target DNA molecule in a homogeneous assay. If the target is not present, only uncorrelated events originating from free probes will be observed at either channel. Phage lambda DNA in a background of salmon genomic DNA was detected as a two-dye coincident signal at a relative concentration of one lambda molecule per salmon genome. In a control sample, cleavage of the lambda DNA between the two probe binding sites eliminated the coincident signals. In a second experiment, a single-copy transgene was detected in maize. Detection parameters and possible future applications to genetic analysis are discussed.

Animals↗
PubMed1997

Newborn genetic identification: a protocol using microsatellite DNA as an alternative to footprinting.

Newborn identification by foot- or finger-printing presents serious drawbacks. This study proposes an alternative method based on DNA analysis of blood-spots taken from the newborn child. CSF1PO, TPOX and TH01 microsatellite loci were chosen to develop a fast and reliable protocol to be applied in cases where it is suspected that newborn children have been exchanged. The advantage of these loci is that one can simultaneously amplify them by PCR multiplex reaction and determine their alleles, thereby reducing the time needed for identification tests. Moreover, the amplification products of these loci are very small (< 350 bp) and so can be analyzed in samples with degraded DNA. We have been able to prove that it is possible to obtain results in blood-spots taken from newborns up to 13 years before and kept at room temperature. Thus the protocol proposed here can be applied in long-term post-natal identification cases.

Base Sequence↗