Biomedical subjects

G Castillo

Publications and source records attributed to G Castillo.

At least 19 recordsLinked to original sources

PubMed2000

Development and application of a synthetic peptide-based osteocalcin assay for the measurement of bone formation in mouse serum.

The mouse is frequently used as an animal model to study skeletal mechanisms relevant to humans. Biochemical markers of bone formation and resorption provide one of the key parameters for assessing skeletal metabolism. One biochemical marker that has proven to be useful in the studies of mouse skeletal metabolism is osteocalcin. Assay for osteocalcin is available in the mouse. The present study describes development of an osteocalcin radioimmunoassay (RIA) using a synthetic peptide. Intact osteocalcin purified from mouse bone extracts shows parallel displacement with synthetic peptide. Sensitivity of the RIA was 19 ng/ml. The average (n = 9) intra- and interassay coefficient of variation for two controls was less than 10%; the averaged recoveries were 106%. The osteocalcin concentration measured by peptide RIA shows a high correlation (r = 0.88, n = 117, P < 0.0001) with an intact osteocalcin assay. In addition, when the intact assay and peptide assays were applied to evaluate skeletal perturbation, similar results were obtained. Accordingly, osteocalcin levels measured by both intact and peptide-based RIA in 8-week C57BL/6J (n = 8) mice treated with PTH 1-34 were twofold higher compared with the vehicle-treated control group. Further studies of the application of the peptide-based RIA for osteocalcin revealed that osteocalcin levels in 4-week postovariectomized (OVX) C57BL/6N mice (n = 10) were 80% higher than the sham-operated (n = 10) mice receiving vehicle. OVX mice receiving weekly injections of estradiol (400 microg/kg body weight) were 38% lower compared with the OVX group treated with vehicle. In conclusion, the peptide-based RIA has analytical and a discriminative power similar to that of the intact osteocalcin assay but has the advantage that the resources for this assay are much easier to accrue.

Animals↗

PubMed2000

Development and application of a serum C-telopeptide and osteocalcin assay to measure bone turnover in an ovariectomized rat model.

Biochemical markers applicable to the ovariectomized rat model can provide important tools for studying the bone remodeling process in this animal model of postmenopausal osteoporosis. We describe the development and application of two biochemical markers, a C-telopeptide (of type-I collagen) enzyme-linked immunosorbent assay (ELISA) for measuring bone resorption and an osteocalcin radioimmunoassay (RIA) for measuring bone formation in rat serum. The C-telopeptide ELISA is based on an affinity purified polyclonal antibody generated against human sequence DFSFLPQPPQEKAHDGGR. The antibody epitope involves amino acid sequence, which is similar in rat and human carboxyl terminal peptide of type-I (alpha 1) collagen. Sensitivity of the ELISA was 0.3 ng/ml. The averaged intra- and interassay variation was CV <7%. Averaged dilution and spiked recoveries were 91% and 105%, respectively. The second marker developed is a synthetic peptide-based osteocalcin RIA, which does not require isolation and purification of intact osteocalcin from rat bone. Osteocalcin antiserum used in the RIA was generated in rabbits against a synthetic peptide comprising amino acids 33-49 of the rat osteocalcin sequence. The sensitivity of the RIA was 0.15 ng/ml of peptide. The averaged intra (n = 10) and interassay variations for two controls were CV <9% and 12%, respectively. The averaged dilution and spiked recoveries were 99.6%. In vivo validation of the C-telopeptide ELISA and osteocalcin RIA was performed in an ovariectomized (OVX) rat model. In 12-week-old OVX Sprague Dawley rats, the C-telopeptide and osteocalcin concentrations were approximately 65% and 40%, respectively, higher than the sham group. Estradiol repletion significantly lowered the C-telopeptide and osteocalcin concentration to the levels of the sham group. In addition, changes in serum C-telopeptide concentration correlated negatively with trabecular BMD measured by pQCT (r = -0.51, P < 0.001). In conclusion, the C-telopeptide ELISA and osteocalcin RIA exhibited required sensitivity, accuracy, and adequate discriminatory power to be used for measuring bone resorption and bone formation in the ovariectomized rat model.

Amino Acid Sequence↗

PubMed2000

Development and evaluation of C-telopeptide enzyme-linked immunoassay for measurement of bone resorption in mouse serum.

The mouse is increasingly being used as an animal model for the study of skeletal phenotypes in humans, mainly because of the ease of genetic manipulation. Biochemical markers of bone metabolism provide a valuable parameter for the assessment of skeletal metabolism. In the mouse model, assays for bone formation have been available for a long time; however, little is known about bone resorption markers. The present study describes the development of a serum C-telopeptide enzyme-linked immunoassay (ELISA), which measures degradation products of type I collagen that are generated by osteoclastic bone resorption. The C-telopeptide ELISA uses affinity-purified antibodies generated against human sequence DFSFLPQPPQEKAHDGGR. The epitope involves an amino acid sequence, which is identical in the mouse and human C-terminal peptide of type I collagen (alpha1 chain). Sensitivity of the ELISA used was <0.1 ng/mL. The average intra- (n = 10) and interassay (n = 8) coefficient of variation for two controls was <12%. The average dilution and spike recovery rates were 98% and 97%, respectively. Application of the ELISA to measure C-telopeptide in 3-4-week postovariectomized (ovx) C57BL/6J (B6) mice (n = 9 or 10) showed a 45% higher C-telopeptide concentration than the sham-operated mice. Treatment of ovx mice with estradiol (400 microg/kg body weight) or alendronate (1.0 mg/kg body weight) resulted in a 20%-50% decrease in C-telopeptide levels compared to the vehicle-treated ovx group. In addition, B6 mice fed a calcium-deficient diet (0.01% calcium) showed a 50% higher C-telopeptide concentration compared to the B6 mice receiving a normal diet (0.6% calcium). In conclusion, the C-telopeptide ELISA exhibited acceptable analytical performance and sufficient discriminatory power to show expected directional changes in the rate of bone resorption following ovariectomy, ovx plus estradiol or alendronate treatment, and administration of a calcium-deficient diet. Therefore, the ELISA developed in this study could be used for measuring bone resorption in the mouse model.

Alendronate↗

PubMed1999

An adipogenic cofactor bound by the differentiation domain of PPARgamma.

Ligand activation of the nuclear receptor PPARgamma induces adipogenesis and increases insulin sensitivity, while activation of other PPAR isoforms (-alpha and -delta) induces little or no fat cell differentiation. Expression and activation of chimeras formed between PPARgamma and PPARdelta in fibroblasts has allowed us to localize a major domain of PPARgamma responsible for adipogenesis to the N-terminal 138 amino acids, a region with AF-1 transcriptional activity. Using this region of PPARgamma as bait, we have used a yeast two-hybrid screen to clone a novel protein, termed PGC-2, containing a partial SCAN domain. PGC-2 binds to and increases the transcriptional activity of PPARgamma but does not interact with other PPARs or most other nuclear receptors. Ectopic expression of PGC-2 in preadipocytes containing endogenous PPARgamma causes a dramatic increase in fat cell differentiation at both the morphological and molecular levels. These results suggest that interactions between PGC-2, a receptor isoform-selective cofactor and PPARgamma contribute to the adipogenic action of this receptor.

Adipocytes↗

PubMed1999

Madangolide and laingolide A, two novel macrolides from lyngbya bouillonii (Cyanobacteria)

Two new macrolide derivatives, madangolide (2) and laingolide A (3), have been isolated from the cyanobacterium Lyngbya bouillonii, collected in Papua New Guinea. Their structures (without stereochemistry) have been established by detailed high-field 1D and 2D NMR studies and, in the case of 3, by comparison with the spectroscopic data of laingolide (1), previously isolated from the same organism.

Journal Article↗

PubMed1999

Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase.

A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100-1000-fold less efficiently. The best kcat/Km values were found with benzaldehyde > 3-methoxybenzaldehyde > octanal > coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.

Alcohol Dehydrogenase↗

PubMed1999

Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator toxT in response to culture conditions.

Vibrio cholerae El Tor require special in vitro culture conditions, consisting of an initial static growth period followed by shift to shaking (AKI conditions), for expression of cholera toxin (CT) and toxin coregulated pili (TCP). ToxT, a regulator whose initial transcription depends on the ToxR regulator, positively modulates expression of CT and TCP. To help understand control of CT and TCP in El Tor vibrios, we monitored ctxAB and ToxR-dependent toxT transcription by time course primer extension assays. AKI conditions stimulated CT synthesis with an absence of ctxAB transcription during static growth followed by induction upon shaking. ToxR-dependent toxT transcription was induced at the end of the static growth period but was transient, stopping shortly after shaking was initiated but, interestingly, also if the static phase was prolonged. Immunoblot assays showed that ToxR protein levels were not coincidentally transient, implying a protein on/off switch mechanism for ToxR. Despite the transient activation by ToxR, transcription of ctxAB was maintained during shaking. This finding suggested continued toxT expression, possibly through relay transcription from another promoter. The 12.6-kb distant upstream tcpA promoter responsible for expression of the TCP operon has been proposed to provide an alternate toxT message by readthrough transcription. Activation of the tcpA promoter is supported by increased expression of TcpA protein during the shaking phase of the culture. Readthrough transcription of toxT from tcpA would be compatible with reverse transcription-PCR evidence for a toxT mRNA at times when ToxR-dependent transcription was no longer detectable by primer extension.

Bacterial Outer Membrane Proteins↗

PubMed1998

Resistance to antibiotic and heavy metals of motile aeromonads from Chilean freshwater.

In this work the resistance of 172 motile Aeromonas isolates recovered from raw drinking water supplies (56), irrigation waters (60) and runoff waters receiving sewage (56), to some antibiotics and heavy metals was investigated by agar diffusion and agar dilution methods. A high proportion of isolates from all water sources showed resistance to carbenicillin, erythromycin, streptomycin, cephradine and cadmium, and susceptibility to chloramphenicol, kanamycin, gentamicin, tetracycline, nalidixic acid, trimethoprim-sulphametoxazole and chromium. No amikacin-resistant Aeromonas were recovered. No relationship was found between antimicrobial resistance and Aeromonas species, with the exception of cephradine, that exhibited a significantly higher activity against the A. sobria isolates than the other Aeromonas species (P < 0.05). Moderately polluted waters showed lower antibiotic multiresistance and metal susceptibility than unpolluted and highly polluted ones. Although significant differences (P < 0.05), between resistance frequencies to erythromycin, carbenicillin, streptomycin and cephradine were found among isolates from different sources, the antimicrobial resistance patterns of aeromonads could not be related to the level of faecal pollution. These results indicate that aeromonads resistant to antibiotics and heavy metals are easily recovered from water sources in Chile, posing a potential public health risk.

Aeromonas↗

PubMed1998

Cognitive brain activity in Alzheimer's disease: electrophysiological response during picture semantic categorization.

Semantic memory deterioration is a major component of the cognitive decline seen in patients with dementia of the Alzheimer's type (DAT); however, the exact nature of this deficit remains unclear. Some research data support a procedural deficit where there is an inability to access or retrieve the contents of semantic memory, while other data point to a degraded semantic store where the actual content of semantic memory is degraded. Additional information about semantic processing in DAT can be obtained through the use of an event-related potential (ERP) component known as N400. In the present study, ERPs were recorded from 10 young control participants, 10 elderly control participants, and 10 DAT patients in a picture-semantic matching task. Stimuli were presented sequentially as prime-target pairs, with one-half of the targets matching the primes via semantic relationships (e.g., piano-violin) and the other half mismatching the prime (e.g., helmet-violin). The task was to discriminate between semantically related and unrelated pairs of pictures. In the young and elderly control groups, ERPs generated a larger N400 for unrelated than related target pictures, with a maximum amplitude around 380 ms in the young group and around 480 ms in the elderly group. The amplitude of the N400 was significantly reduced in the DAT patients. However, a separate analysis of congruent and incongruent ERPs trials revealed significant differences only with the incongruent trials. The amplitude of incongruent recordings was larger for the elderly control group than for the DAT patients, while the amplitude for congruent recordings was similar in both groups. These findings are consistent with the neuropathological evidence that Alzheimer's disease is a neocortical disconnection syndrome in which there is a loss of structural and functional integrity of long corticocortical tracts. The semantic activation created by the context is not used efficiently in processing stimuli, which affects access to specific concepts and gradually leads to a breakdown in the structure and organization of semantic memory.

Adult↗

PubMed1997

Down-regulation of the mdr gene by thyroid hormone during Xenopus laevis development.

The developmental regulation of mdr in Xenopus laevis has been investigated. Xe-mdr expression was first detected in the early tadpole stage just prior to the onset of feeding and increased during intestinal development, with a sharp decline at metamorphosis. Xe-mdr expression was found to be localized specifically to the epithelial cells lining the intestinal tract. When premetamorphic tadpoles were treated with 5 nM triiodothyronine to induce metamorphosis, a significant decrease in mdr message and protein was observed after 3 days, a time at which the primary epithelium remained intact. Furthermore, in thyroid-hormone treated primary cultures of brush border epithelial cells, a reduction in mdr message also was observed. These results demonstrate that the Xe-mdr gene is developmentally regulated and suggest a role for thyroid hormone in this process. This is the first report of a naturally occurring substance that can down-regulate mdr gene expression in vivo.

Animals↗

PubMed1997

Opioid availability in Latin America: the Santo Domingo report progress since the Declaration of Florianopolis.

The World Health Organization (WHO) has indicated that opioid analgesics are insufficiently available, particularly in developing countries, due to a variety of reasons, including legislative, educational, and policy issues. In its effort to promote the rational use of medical opioids and the adequate treatment of patients with cancer, WHO has sponsored a meeting of Latin American representatives every 2 years, which includes health professionals and government regulators. During March 24-27, 1996, a group of 86 representatives of cancer pain relief and palliative care programs from nine Latin American countries met in Santo Domingo under the auspices of the WHO Palliative Care Program for Latin America. For the first time since the First Latin American Meeting, government regulators were present to help address the issue of opioid availability from their perspective. During the meeting, issues pertaining to cancer pain, opioid availability, and palliative care were discussed. This report summarizes some of the events and presents a summary of the conclusions of an earlier meeting in 1994, as described in the Declaration of Florianopolis, and presents its follow-up, The Santo Domingo Report, generated following the 1996 meeting.

Analgesics, Opioid↗

PubMed1996

Isocratic high-performance liquid chromatographic method for quantitative determination of lysine, histidine and tyrosine in foods.

A method for the quantitative determination of lysine, histidine and tyrosine in foods based on pre-column derivatization with 5-dimethylaminonaphthalene-1-sulfonyl chloride (DnsCl) and reversed-phase liquid chromatography has been developed. Derivatization conditions, including DnsCl concentration, time, temperature, and buffer solution were studied. To establish the reliability of the proposed liquid chromatographic (LC) method, the precision and accuracy of the analyses were evaluated using samples of casein and lysozyme.

Caseins↗

PubMed1996

Amebiasis of the penis.

Penile amebiasis is rarely a recognized entity because the penis is not the usual site of presentation for amebiasis. Homosexual men have a higher risk of acquiring the lesion. Amebic ulcers resemble cutaneous lesions arising from squamous cell carcinoma, chancroid, primary syphilis, granuloma inguinale, and many other causes. An amebic ulcer should be suspected in a patient with balanoposthitis that resists antibiotic therapy. Biopsy is fundamental to isolate the trophozoites to confirm the diagnosis. Metronidazole and hydrochloric emetine are still the treatment of choice. This diagnosis should especially be considered in cases of lesions detected in patients who practice anogenital sex or who are immunocompromised.

Adolescent↗

PubMed1995

A homologue of the mammalian multidrug resistance gene (mdr) is functionally expressed in the intestine of Xenopus laevis.

P-glycoprotein is an integral membrane protein that functions in multidrug resistance (MDR) cells as a drug efflux pump to maintain intracellular concentrations of antitumor drugs below cytotoxic levels. A homologue of the mammalian mdr gene has been isolated and characterized from Xenopus laevis (Xe-mdr). The cDNA was isolated from a tadpole cDNA library using the full length mouse mdrlb cDNA as a probe. The Xe-mdr encodes a protein that is 66% identical to the mouse mdrlb and 68% identical to the human mdrl. The predicted structure of the Xe-mdr gene product identifies twelve membrane spanning domains and two ATP binding sites both of which are the hallmark of the ABC (ATP binding cassette) transporters. Xe-mdr mRNA is expressed as a single message of 4.5 kb and is found predominantly in the intestine. Xe-mdr message is increased 3- to 4-fold in the ileum compared to the rest of the small intestine. In situ hybridization of sequential sections from the small intestine localized the expression of the Xe-mdr to the cells lining the lumenal epithelium. Brush border membrane vesicles prepared from the small intestine of Xenopus laevis effluxed vinblastine in an ATP-dependent manner. Efflux was decreased by verapamil, a known inhibitor of P-glycoprotein function. These studies indicate that the structure of Xe-mdr has been conserved and suggest that the protein has a role in maintaining the function of the normal intestine in Xenopus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1995

Inhibitory effect of sodium ursodeoxycholate on basal and stimulated short-circuit current across the isolated toad skin.

The effect of sodium ursodeoxycholate (U) on short-circuit current (SCC), an index of basal and stimulated net ion transport across isolated skins of Bufo arenarum toads, was tested. U inhibited basal SCC when added to the epidermal side of the skins. The inhibitory effect was reversible after rinsing the preparation during 60 min. U also inhibited the natriferic response to oxytocin, db-cAMP and theophylline by 82%, 49% and 47%, respectively. Inhibition of SCC by exposure to U was reversed by the polyene antibiotic nystatin. In turn, SCC induced by nystatin in the amiloride-treated skin was insensitive to U and blocked by ouabain, a Na+, K(+)-ATPase inhibitor. These results strongly suggest that the effect of U is exerted at the apical membrane of sodium transporting cells, and rule out the existence of an additional site of inhibitory action of U.

Action Potentials↗

PubMed1995

Opioid availability in Latin America: the declaration of Florianopolis.

Between March 27 and 29, 1994, a group of representatives of 32 palliative care programs from eight Latin American countries met under the auspices of the World Health Organization (WHO) Palliative Care Program for Latin America in Florianopolis, Brazil. The participants included physicians, nurses, psychologists, volunteers, drug regulators, hospital administrators, and representatives from the pharmaceutical industry. A comprehensive report by David Joranson (University of Wisconsin-Madison) was followed by a general discussion moderated by Jan Stjernsward, Chief of the Cancer Unit, World Health Organization, and by Eduardo Bruera, Coordinator of the WHO Cancer Pain and Palliative Care Program for Latin America. A number of issues related to opioid availability were identified and discussed. This declaration summarizes the main conclusions of the meeting. The attendants would like to encourage the widest possible distribution of this document.

Analgesics, Opioid↗

PubMed1995

Metabolic inhibition and chloride transport in isolated toad skin.

1. When added to the Na(+)-containing solution bathing the isolated toad skin, dinitrophenol (DNP, an uncoupler of oxidative phosphorilation) caused decreases in the baseline values of short circuit current (SCC) and transepithelial conductance (G). 2. DNP also inhibited the increases in SCC and G caused by theophylline, whether added prior to the xanthine, or after the effect of the latter was fully developed. 3. In skins exposed to theophylline and bathed in Cl(-)-free (sulfate Ringer's) solution, the changes in SCC and G had a similar time course (t1/2 > 15 min). In the presence of Cl- (skins bathed in Ringer's solution), SCC decreased with a similar rate, whereas the rate of the decrease in G was greater (t1/2 < 15 min). 4. DNP also decreased the SCC induced by a Cl- concentration gradient in skins exposed to theophylline (SCCg) with a time course similar to its effect on the theophylline-increased G in the presence of Cl-. DNP was effective irrespective of the presence of ambient Na+. 5. A similar difference was observed in skins bathed in CIR and exposed to forskolin. In contrast to theophylline, however, forskolin partially overcame the inhibition of G brought about by DNP; no such recovery was observed in SCC. 6. In contrast to its influence on the responses to theophylline and forskolin, DNP failed to prevent either the increase in G or the onset of SCCg in skins exposed to dibutyryl cyclic AMP. 7. Rotenone, an inhibitor of the electron-transport chain, significantly decreased SCC and G in the unstimulated skin. It also prevented the SCC response to theophylline, and decreased it if added after the effects of the xanthine were fully developed, but failed to modify the increase in G brought about by theophylline. The time course of SCC inhibition by rotenone was similar to that caused by DNP. 8. Ouabain, an inhibitor of Na+,K(+)-ATPase, decreased SCC in the theophylline-stimulated skin, without affecting G. 9. We conclude that, whereas integrity of oxidative energy metabolism is necessary to sustain SCC in the isolated toad skin, it is not a strict requirement for the increase of Cl(-)-dependent G activated by cAMP. 10. The effect of DNP on Cl(-)-dependent G activated by cAMP is probably exerted at the cAMP generation step, by inhibition of adenyl cyclase and/or a decrease in the availability of ATP.

Animals↗

PubMed1995

[Comparison between digoxin and atenolol in chronic atrial fibrillation].

The benefits of digoxin in patients with atrial fibrillation may be reduced due to its limited effect on atrioventricular conduction. The aim of this work was to compare digoxin and atenolol on functional class, resting and exercise heart rate and exercise capacity in patients with atrial fibrillation. Thirteen subjects with this condition, normal echocardiographic left ventricular function and size, a resting heart rate less than 80 beats/min and with no contraindication for beta blocker or digoxin use were studied. Patients were randomly assigned to receive initially digoxin 0.25 mg o.d. or atenolol 100 mg o.d. in a double blind fashion. The doses were adjusted to obtain a heart rate between 60 and 80 beats/min at the end of the first week of treatment. After two weeks of treatment, outcomes were assessed, patients were left without treatment for one week and crossed over to the other drug after that. Resting heart rates achieved with digoxin and atenolol were similar (67 +/- 11 and 65 +/- 23 beats/min respectively). However, maximal exercise heart rates and maximal exercise time were higher during digoxin treatment (166 +/- 23 vs 135 +/- 27 beats/min and 9.95 +/- 1.68 vs 8.5 +/- 2 min respectively). NYHA functional class deteriorated in three patients receiving atenolol. We conclude that atenolol achieves a better control of heart rate during exercise but also reduces maximal exercise capacity.

Adult↗