Biomedical subjects

R Sandoval

Publications and source records attributed to R Sandoval.

At least 19 recordsLinked to original sources

PubMed2001

Ca(2+) signalling and PKCalpha activate increased endothelial permeability by disassembly of VE-cadherin junctions.

1. The role of intracellular Ca(2+) mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) in both cases was due to release of Ca(2+) from intracellular stores and influx of extracellular Ca(2+). 2. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial (125)I-albumin permeability. Thapsigargin induced activation of PKCalpha and discontinuities in VE-cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCalpha activation and similar alterations in VE-cadherin junctions, but in association with actin stress fibre formation. 3. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC(20)), whereas thrombin induced MLC(20) phosphorylation consistent with formation of actin stress fibres. 4. Calphostin C pretreatment prevented the disruption of VE-cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca(2+)](i) elicited by thapsigargin and thrombin may activate a calphostin C-sensitive PKC pathway that signals VE-cadherin junctional disassembly and increased endothelial permeability. 5. Results suggest a critical role for Ca(2+) signalling and activation of PKCalpha in mediating the disruption of VE-cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

Albumins↗

PubMed2001

Requirement for Ca2+ signaling in the mechanism of thrombin-induced increase in endothelial permeability.

We compared the thrombin-activated responses in human umbilical vein endothelial cells (HUVECs) and a HUVEC-derived cell line, ECV304. Thrombin induced a 40-50% decrease in transendothelial monolayer electrical resistance and a twofold increase in 125I-albumin permeability in HUVECs, whereas it failed to alter the endothelial barrier function in ECV304 cells. Thrombin produced a brisk intracellular Ca2+ concentration transient and phosphorylation of 20-kDa myosin light chain in HUVECs but not in ECV304 cells. Thrombin-induced phosphoinositide hydrolysis was comparable in ECV304 cells and HUVECs, indicating the activation of thrombin receptors in both cell types. La3+ reduced both the thrombin-induced decrease in endothelial monolayer electrical resistance and the increase in 125I-albumin permeability in HUVECs. Because the absence of Ca2+ signaling could explain the impairment in the permeability response in ECV304 cells, we studied the effect of increasing intracellular Ca2+ concentration in ECV304 cells with thapsigargin. Exposure of ECV304 cells to thapsigargin caused decreased endothelial monolayer electrical resistance and increased 125I-albumin permeability. These results indicate that Ca2+ influx and activation of Ca2+-dependent signaling pathways are important determinants of the thrombin-induced increase in endothelial permeability.

Antigens, CD↗

PubMed2001

Synergistic effects of tumor necrosis factor-alpha and thrombin in increasing endothelial permeability.

Because activation of the coagulation cascade and the generation of thrombin coexist with sepsis and the release of tumor necrosis factor (TNF)-alpha, we determined the effects of TNF-alpha on the mechanism of thrombin-induced increase in endothelial permeability. We assessed Ca(2+) signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cells exposed to TNF-alpha for 2 h, thrombin produced a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)) lasting up to 10 min. In contrast, thrombin alone produced a rise in [Ca(2+)](i) lasting for 3 min, whereas TNF-alpha alone had no effect on [Ca(2+)](i.) Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-alpha-exposed cells. In the absence of extracellular Ca(2+), thrombin produced similar increases in [Ca(2+)](i) in both control and TNF-alpha-exposed cells. In TNF-alpha-exposed cells, the thrombin-induced Ca(2+) influx after intracellular Ca(2+) store depletion was significantly greater and prolonged compared with control cells. Increased Ca(2+) entry was associated with an approximately fourfold increase in Src activity and was sensitive to the Src kinase inhibitor PP1. After TNF-alpha exposure, thrombin caused increased tyrosine phosphorylation of junctional proteins and actin stress fiber formation as well as augmented endothelial permeability. These results suggest that TNF-alpha stimulation of endothelial cells results in amplification of the thrombin-induced Ca(2+) influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function.

Albumins↗

PubMed2000

Molecular characterization of human immunodeficiency virus type 1-infected individuals from bolivia reveals the presence of two distinct genetic subtypes B and F.

Thirty HIV-1-positive samples from Bolivia were genetically characterized on the basis of HMA and DNA sequencing, revealing the presence of B and F subtypes, in accordance with the molecular epidemiology pattern already described for other South American countries such as Brazil and Argentina. The interpatient divergence of subtype B Bolivian specimens was on average 14.2% (4.3-19.8%) at the nucleotide level, whereas the two unlinked subtype F samples (BO23 and BO29) were only 8.2% divergent, suggesting a more recent introduction of this subtype in the country. In our study group, which represents 13% of the HIV/AIDS cases already described in Bolivia as of May 1996, the transmission occurred more frequently through heterosexual exposures (46.7%), followed by homosexual (23.3%), bisexual (10%), intravenous drug use (3.3%), and vertical (3.3%); in one case the potential exposure category could not be defined (3.3%). No association could be established between exposure categories, gender, or clinical classification and subtype distribution in the Bolivian HIV/AIDS patients.

Adult↗

PubMed2000

G protein-coupled receptor kinase-5 regulates thrombin-activated signaling in endothelial cells.

We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca(2+)](i). GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca(2+)](i) in response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.

Cells, Cultured↗

PubMed2000

Reversibility of increased microvessel permeability in response to VE-cadherin disassembly.

We determined the role of vascular endothelial (VE)-cadherin complex in regulating the permeability of pulmonary microvessels. Studies were made in mouse lungs perfused with albumin-Krebs containing EDTA, a Ca(2+) chelator, added to study the VE-cadherin junctional disassembly. We then repleted the perfusate with Ca(2+) to restore VE-cadherin integrity. Confocal microscopy showed a disappearance of VE-cadherin immunostaining in a time- and dose-dependent manner after Ca(2+) chelation and reassembly of the VE-cadherin complex within 5 min after Ca(2+) repletion. We determined the (125)I-labeled albumin permeability-surface area product and capillary filtration coefficient (K(fc)) to quantify alterations in the pulmonary microvessel barrier. The addition of EDTA increased (125)I-albumin permeability-surface area product and K(fc) in a concentration-dependent manner within 5 min. The permeability response was reversed within 5 min after repletion of Ca(2+). An anti-VE-cadherin monoclonal antibody against epitopes responsible for homotypic adhesion augmented the increase in K(fc) induced by Ca(2+) chelation and prevented reversal of the response. We conclude that the disassembled VE-cadherins in endothelial cells are mobilized at the junctional plasmalemmal membrane such that VE-cadherins can rapidly form adhesive contact and restore microvessel permeability by reannealing the adherens junctions.

Adherens Junctions↗

PubMed2000

[Intracranial mucocele. Report of a case].

Mucocele, usually associated to paranasal sinuses is defined as an abnormal accumulation and retention of mucous secretions. It usually presents with intraorbital or paranasal and rarely with pure neurological symptoms. We report a 41 years old male who presented with a history of supraorbital left headache, diplopia and proptosis. CT scans and magnetic resonance imaging showed an expansive mass in the right frontal sinus. The patient was operated, the presumptive preoperative clinical diagnosis of mucocele was confirmed and the lesion was drained. A postoperative CT scan confirmed the normal expansion of the frontal lobe.

Adult↗

PubMed1999

Thrombin induces proteinase-activated receptor-1 gene expression in endothelial cells via activation of Gi-linked Ras/mitogen-activated protein kinase pathway.

We addressed the mechanisms of restoration of cell surface proteinase-activated receptor-1 (PAR-1) by investigating thrombin-activated signaling pathways involved in PAR-1 re-expression in endothelial cells. Exposure of endothelial cells transfected with PAR-1 promoter-luciferase reporter construct to either thrombin or PAR-1 activating peptide increased the steady-state PAR-1 mRNA and reporter activity, respectively. Pretreatment of reporter-transfected endothelial cells with pertussis toxin or co-expression of a minigene encoding 11-amino acid sequence of COOH-terminal Galphai prevented the thrombin-induced increase in reporter activity. Pertussis toxin treatment also prevented thrombin-induced MAPK phosphorylation, indicating a role of Galphai in activating the downstream MAPK pathway. Expression of constitutively active Galphai2 mutant or Gbeta1gamma2 subunits increased reporter activity 3-4-fold in the absence of thrombin stimulation. Co-expression of dominant negative mutants of either Ras or MEK1 with the reporter construct inhibited the thrombin-induced PAR-1 expression, whereas constitutively active forms of either Ras or MEK1 activated PAR-1 expression in the absence of thrombin stimulation. Expression of dominant negative Src kinase or inhibitors of phosphoinositide 3-kinase also prevented the MAPK activation and PAR-1 expression. We conclude that thrombin-induced activation of PAR-1 mediates PAR-1 expression by signaling through Gi1/2 coupled to Src and phosphoinositide 3-kinase, and thereby activating the downstream Ras/MAPK cascade.

Amino Acid Sequence↗

PubMed1999

Trauma system impact on admission site: a comparison of two states.

OBJECTIVE: To introduce a measure assessing statewide hospital admission patterns for trauma and evaluate its utility in identifying significant admission redistributions in neighboring states as a function of trauma system implementation. METHODS: A retrospective claims database analysis was performed of hospitalized trauma patients in Washington and Oregon. The site of hospitalization and diagnostic information were used to determine an admission rate difference for each hospital: the rate of index patient admissions minus the rate of non-index patient admissions. Mean admission rate differences for Level II and III versus other hospitals in each state were compared as a function of time. RESULTS: A bias in favor of admitting more serious injuries to Level II and III hospitals was seen with trauma system implementation in both states, but it was more pronounced in the Oregon trauma system. CONCLUSION: Assessment of admission rate differences is a helpful method of characterizing the evolution of statewide trauma systems.

Adult↗

PubMed1999

Time course of recovery of endothelial cell surface thrombin receptor (PAR-1) expression.

We studied dynamics of cell surface expression of proteolytically activated thrombin receptor (PAR-1) in human pulmonary artery endothelial cells (HPAEC). PAR-1 activation was measured by changes in cytosolic calcium concentration ([Ca2+]i) and HPAEC retraction response (determined by real-time transendothelial monolayer electrical resistance). [Ca2+]i increase in response to thrombin was abolished by preexposure to 25 nM thrombin for >60 min, indicating PAR-1 desensitization, but preexposure to 25 nM thrombin for only 30 min or to 10 nM thrombin for up to 2 h did not desensitize PAR-1. Exposure to 10 or 25 nM thrombin decreased monolayer electrical resistance 40-60%. Cells preexposed to 10 nM thrombin, but not those preexposed to 25 nM thrombin, remained responsive to thrombin 3 h later. Loss of cell retractility was coupled to decreased cell surface PAR-1 expression as determined by immunofluorescence. Cell surface PAR-1 disappeared upon short-term (30 min) thrombin exposure but reappeared within 90 min after incubation in thrombin-free medium. Exposure to 25 nM thrombin for >60 min prevented rapid cycloheximide-insensitive PAR-1 reappearance. Cycloheximide-sensitive recovery of cell surface PAR-1 expression required 18 h. Therefore, both duration and concentration of thrombin exposure regulate the time course of recovery of HPAEC surface PAR-1 expression. The results support the hypothesis that initial recovery of PAR-1 surface expression in endothelial cells results from a rapidly mobilizable PAR-1 pool, whereas delayed recovery results from de novo PAR-1 synthesis. We conclude that thrombin itself regulates endothelial cell surface PAR-1 expression and that decreased surface expression interferes with thrombin-induced endothelial cell activation responses.

Calcium↗

PubMed1999

Prenatal diagnosis of nonmosaic trisomy 9 in a fetus with severe renal disease.

We report a case of nonmosaic trisomy 9 presenting at 21 weeks of gestation with polycystic, echogenic horseshoe kidney, collapsed bladder, absent amniotic fluid, and intrauterine growth restriction. Color Doppler imaging demonstrated no blood flow signals from renal vessels. Fetal blood sampling confirmed a 47,XX,+9 karyotype, with no evidence of mosaicism, and increased serum beta2-microglobulin levels of 10.7 mg/l, consistent with severe renal failure. A repeat scan at 23 weeks also revealed a dysmorphic face, bilateral microphthalmia, and a cerebellar vermian defect. Follow-up examinations showed progressive growth restriction leading to fetal death at 33 weeks of gestation. This report demonstrates that fetuses with nonmosaic trisomy 9 may present with severe renal abnormalities and confirms that cases seen in the second and third trimesters usually have a dismal outcome.

Adult↗

PubMed1998

Aminoglycoside antibiotics traffic to the Golgi complex in LLC-PK1 cells.

Aminoglycoside antibiotics are known to be internalized via endocytosis and have been associated with subcellular organelle dysfunction; however, the route of intracellular trafficking and their distribution remain largely unknown. To address these questions, a Texas Red conjugate of gentamicin (TRG) was synthesized for dual-labeling experiments with the endoplasmic reticulum, Golgi, and lysosomal markers DiOC6-3, C6-NBD-ceramide, and fluorescent dextrans, respectively. Confocal images were overlaid to determine areas of colocalization. Initial characterization studies of the fluorescent gentamicin analogue revealed that both internalization and accumulation were inhibited by excess unlabeled gentamicin. Furthermore, the fluorescent gentamicin label was colocalized with unlabeled gentamicin, using immunologic techniques. LLC-PK1 cells were exposed to the fluorescent gentamicin in media containing 1 mg/ml labeled gentamicin for 8 h and then either fixed or chased with gentamicin-free media for an additional 16 or 40 h (24 to 48 h total). Studies with fluorescent dextrans revealed rapid intracellular colocalization within the endosomal and lysosomal systems. Neither endoplasmic reticulum nor mitochondrial colocalization could be detected. However, Golgi colocalization was revealed using both confocal and electron microscopic techniques at 8 h of TRG incubation, and continued to be present for an additional 40 h. Protein synthetic rates were quantified and revealed decreased synthesis at the 24-h chase mark. These results suggest that TRG can serve as a fluorescent tracer for aminoglycoside trafficking within cells. The fluorescent marker remained associated with vesicular structures at all times and colocalized with the Golgi apparatus. It is postulated that this early association of gentamicin with the Golgi complex may be an avenue for delivery of aminoglycosides to other intracellular compartments.

3,3'-Diaminobenzidine↗

PubMed1997

Cellular mechanism of aminoglycoside tolerance in long-term gentamicin treatment.

In the rat, nephrotoxicity results from uptake of gentamicin at the apical membrane of proximal tubule (PT) cells. However, during continuous gentamicin treatment, the PT epithelium has been shown to recover. The mechanism(s) of cellular recovery and development of tolerance remains unknown. Therefore, we undertook studies designed to characterize cellular adaptations that occur during long-term gentamicin (LTG) treatment. After 19 days of gentamicin treatment, electron microscopy morphological evaluation revealed cellular recovery with an apparent mild decrease in height and number of microvilli. Enzymatic analysis of LTG PT membranes showed that apical and basolateral membranes had essentially returned to normal. Analysis of apical membrane lipid content revealed persistent statistically significant (P < 0.01) elevations in phosphatidylinositol (PI). In vivo immunogold morphological studies and biochemical studies in LTG rats revealed that endocytosis of gentamicin was selectively reduced, whereas the markers of fluid-phase (horseradish peroxidase) and receptor-mediated (beta2-microglobulin) endocytoses were unaffected or increased. Biochemical analysis showed that, although gentamicin binding to apical membranes isolated from LTG rats increased greater than twofold (P < 0.05) over membranes from untreated rats, in vivo cellular uptake, quantified with [3H]gentamicin, was reduced. Western blot analysis of LTG apical membranes and immunofluorescent staining of perfusion-fixed LTG kidneys showed no change in megalin levels or its apical membrane localization. These data imply that recovery of PT cells from and tolerance to LTG treatment involve a selective inhibition of gentamicin uptake across the apical membrane. They indicate that the mediators of gentamicin endocytosis were affected differently: PI levels increased, whereas megalin levels did not change. We conclude that selective inhibition of gentamicin uptake during LTG treatment is not affected by a reduction in PI or megalin levels. We postulate that trafficking of gentamicin and/or gentamicin-containing endocytic structures is reduced in LTG rats, allowing cells to develop tolerance to gentamicin.

Adaptation, Physiological↗

PubMed1995

Psychometric characteristics of the Wender Utah Rating Scale (WURS): reliability and factor structure for men and women.

The goals of this study were to examine the factor structure of the Wender Utah Rating Scale (WURS), to evaluate potential gender differences in factor composition, and to assess the reliability of the scale. The WURS was completed by 310 fathers and 305 mothers of children referred for evaluation of Attention-Deficit Hyperactivity Disorder (ADHD). For males, a five-factor solution (Conduct Problems, Learning Problems, Stress Intolerance, Attention Problems, Poor Social Skills/Awkward) accounted for 72 percent of the variance. There was also a five-factor solution for females (Dysphoria, Impulsive/Conduct, Learning Problems, Attention and Organizational Problems, Unpopular) which accounted for 71 percent of the variance. Symptoms of inattention and impulsivity loaded on separate factors for both men and for women. Test-retest reliability was examined with a different sample of 57 adults who completed the WURS on two separate occasions, 1 month apart. The WURS demonstrated satisfactory internal consistency and temporal stability, and it may be a useful tool for the study of ADHD in adults.

Adolescent↗

PubMed1995

Mal de Meleda.

A case of mal de Meleda originating in Chile is reported. The patient had no history of consanguinity and cases could not be detected in four generations of the patient's family. The palmoplantar keratoderma was progrediens and transgrediens, with distant hyperkeratotic plaques of the knees and elbows. The nails, hair, and teeth were normal.

Adult↗

PubMed1994

Psychometric properties of the children's atypical development scale.

The Children's Atypical Development Scale (CADS) is a 53-item rating scale designed to measure unusual behaviors in children. Principal-factor analysis on a clinic-referred and pediatric sample of 474 children resulted in a four-factor solution: Communication Deficits, Lability, Social Relatedness Deficits, and Preoccupation. The CADS is internally consistent and has adequate temporal stability. CADS factor scores were differentially associated with parent and teacher rating scales, IQ, and Continuous Performance Test errors. The scale shows promise as a clinical and research tool for assessing atypical behaviors associated with pervasive developmental disorder and other neurobehavioral disorders.

Adolescent↗

PubMed1994

Pigmented follicular cyst.

A pigmented follicular cyst is an infrequent variant of an epithelial cyst which has rarely been reported since it was first described about 10 years ago. The condition consists of a single pigmented lesion which occurs predominantly in adult men, and is usually located on the head and neck region. We report an example of a pigmented follicular cyst, which was located in the axilla.

Adult↗

PubMed1994

[Experience with prostaglandins E2 in intracervical gel and intravaginal tablets in fetal deaths].

Our clinical experience with E2 prostaglandins in intracervical gel and vaginal tablets to induce deliveries or abortions with dead fetuses is presented. The overall success rate of 50 patients was 70%, excluding 9 cases of anencephalos fetuses; the rate is increased to 77.7% in the cases of retained abortions and to 78.3% in the cases of dead fetuses in uterus after 20 weeks of gestation.

Abortion, Induced↗