RESUMEN
Eleven pregnant pony mares (D270-326) were administered ceftiofur sodium intramuscularly at 2.2 mg/kg (n = 6) or 4.4 mg/kg (n = 5), once daily. Plasma was obtained prior to ceftiofur administration and at 0.5, 1, 2, 4, 8, 12, and 24 hr after administration. Eight pony mares were re-enrolled in the study at least 3 days from expected foaling to ensure steady-state concentrations of drug at the time of foaling. Mares were administered ceftiofur sodium (4.4 mg/kg, IM) daily until foaling. Parturition was induced using oxytocin 1 hr after ceftiofur sodium administration. Allantoic and amniotic fluid, plasma, and colostrum samples were collected at time of foaling. Serial foal plasma samples were obtained. Placental tissues were collected. Desfuroylceftiofur acetamide (DCA) concentrations were measured in samples by high-performance liquid chromatography (HPLC). Mean (±SD) peak serum concentrations of DCA were 3.97 ± 0.50 µg/ml (low dose) and 7.45 ± 1.05 µg/ml (high dose). Terminal half-life was significantly (p = .014) shorter after administration of the low dose (2.91 ± 0.59 hr) than after administration of the high dose (4.10 ± 0.72 hr). The mean serum concentration of DCA from mares at time of foaling was 7.96 ± 1.39 µg/ml. The mean DCA concentration in colostrum was 1.39 ± 0.70 µg/ml. DCA concentrations in allantoic fluid, amniotic fluid, placental tissues, and foal plasma were below the limit of quantification (<0.1 µg/ml) and below the minimum inhibitory concentration of ceftiofur against relevant pathogens. These results infer incomplete passage of DCA across fetal membranes after administration of ceftiofur sodium to normal pony mares.
Asunto(s)
Antibacterianos/farmacocinética , Cefalosporinas/farmacocinética , Alantoides/química , Líquido Amniótico/química , Animales , Antibacterianos/administración & dosificación , Antibacterianos/análisis , Antibacterianos/sangre , Cefalosporinas/administración & dosificación , Cefalosporinas/análisis , Cefalosporinas/sangre , Calostro/química , Femenino , Feto/química , Semivida , Caballos/metabolismo , Inyecciones Intramusculares/veterinaria , Trabajo de Parto Inducido/veterinaria , Placenta/química , Embarazo/metabolismoRESUMEN
BACKGROUND: PRDM1 is a transcriptional repressor that contributes to primordial germ cell (PGC) development. During early gastrulation, epiblast-derived PRDM1 is thought to be restricted to a lineage-segregated germ line in the allantois. However, given recent findings that PGCs overlap an allantoic progenitor pool that contributes widely to the fetal-umbilical interface, posterior PRDM1 may also contribute to soma. RESULTS: Within the posterior mouse gastrula (early streak, 12-s stages, embryonic days â¼6.75-9.0), PRDM1 localized to all tissues containing putative PGCs; however, PRDM1 was also found in all three primary germ layers, their derivatives, and two presumptive growth centers, the allantoic core domain and ventral ectodermal ridge. While PRDM1 and STELLA colocalized predominantly within the hindgut, where putative PGCs reside, other colocalizing cells were found in non-PGC sites. Additional PRDM1 and STELLA cells were found independent of each other throughout the posterior region, including the hindgut. The Prdm1-Cre-driven reporter supported PRDM1 localization in the majority of sites; however, some Prdm1 descendants were found in sites independent of PRDM1 protein, including allantoic mesothelium and hindgut endoderm. CONCLUSIONS: Posterior PRDM1 contributes more broadly to the developing fetal-maternal connection than previously recognized, and PRDM1 and STELLA, while overlapping in putative PGCs, also co-localize in several other tissues. Developmental Dynamics 246:50-71, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Gástrula/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva/análisis , Alantoides/química , Animales , Proteínas Cromosómicas no Histona , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Germinales Embrionarias , Endodermo/química , Endodermo/embriología , Femenino , Feto/metabolismo , Gástrula/citología , Ratones , Placenta/metabolismo , Embarazo , Proteínas Represoras/análisisRESUMEN
The interaction between prenatal environments and postnatal environments is an important source of phenotypic variability. We examined the ability of prenatal steroid exposure and postnatal energy restriction to explain adrenocortical function and fledging age in captive seabird chicks. We proposed and tested two hypotheses: (1) the strength of prenatal effects is attenuated by challenging postnatal environments (postnatal override) and (2) the strength of prenatal effects increases with the severity of postnatal challenges (postnatal reveal). We reared common murre (Uria aalge) chicks and measured prenatal exposure to corticosterone (CORT) and testosterone (T) from allantoic waste. Adrenocortical function was assessed after 10 d of ad lib. feeding and then after 5 and 10 d on controlled diets. Postnatal override predicts that prenatal steroids will explain more phenotypic variation before implementation of energy restriction; postnatal reveal predicts that the contribution of prenatal steroids will increase with duration and severity of energy restriction. Energy restriction increased secretion of baseline CORT and the adrenocortical response to the standardized stressor of handling and restraint. The ability of prenatal steroids to explain baseline CORT increased with duration of energy restriction, and for day 20 free baseline CORT, there was a significant interaction between kilojoules per day and prenatal CORT levels; severity of restriction strengthened the relationship between prenatal hormone levels and postnatal hormone levels. Both maximum CORT at day 20 and fledging age were best explained by diet treatment and day 15 or day 20 baseline CORT, respectively. Overall, prenatal CORT increased fledging age and baseline secretion of CORT, while prenatal T decreased them. However, prenatal effects on adrenocortical function were apparent only under the energy restriction conditions. Thus, we found some support for the postnatal reveal hypothesis; our results suggest that some prenatal effects on phenotype may be more likely to manifest in challenging postnatal environments.
Asunto(s)
Charadriiformes/embriología , Charadriiformes/crecimiento & desarrollo , Corticosterona/análisis , Corticosterona/sangre , Estrés Fisiológico/fisiología , Testosterona/análisis , Testosterona/sangre , Corteza Suprarrenal/fisiología , Alantoides/química , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Charadriiformes/metabolismo , Metabolismo Energético , Femenino , Masculino , Fenotipo , Restricción Física/fisiologíaRESUMEN
This study was designed to characterize development of the ovine conceptus throughout gestation to establish the temporal relationships in metabolites, electrolytes, fluid volumes within the placenta, and hormonal changes with fetal growth. Length and weight of placentae, weight of cotyledons, and uterine weight increased between d 25 and 80 of gestation in advance of increases in fetal growth between d 80 and 140 of gestation. Allantoic fluid volumes changed (P < 0.01) between d 25 (21 mL) and 40 (91 mL), decreased to d 70 (32 mL), and then increased to d 140 (438 mL). Concentrations and total amounts of proteins in allantoic fluid were reduced between d 25 and 50, but total protein increased (P < 0.01) from d 40 (63 mg) to d 140 (2,991 mg). Concentrations of fructose in allantoic fluid varied between 2 and 6 mg/mL throughout gestation, but total fructose increased (P < 0.01) between d 25 (46 mg) and d 120 (679 mg). Concentrations of glucose ranged from 0.1 to 0.3 mg/mL, and total glucose increased (P < 0.05) from d 25 (3 mg) to d 140 (63 mg) of gestation. Amniotic fluid volume increased (P < 0.01) between d 30 and 140. Concentrations of estrogens in allantoic fluid, maternal uterine artery, and uterine vein increased (P < 0.01) with advancing pregnancy, and concentrations of progesterone in allantoic fluid (P < 0.07) and plasma (P < 0.05) were affected by day of gestation. Concentrations of glucose were greater (P < 0.05) in uterine artery than uterine vein, but concentrations of electrolytes and osmolarity of plasma were not affected by day of gestation. Increases in weights of fetal organs were proportional to increases in fetal weight during gestation. Results of the present study of conceptus growth and development highlight areas of needed research and provide benchmarks for comparisons when evaluating effects of various treatments, environmental conditions, and epigenetics on successful outcomes of pregnancy in sheep.
Asunto(s)
Desarrollo Fetal , Feto/fisiología , Preñez , Oveja Doméstica/embriología , Oveja Doméstica/fisiología , Alantoides/química , Animales , Femenino , Florida , Hormonas Esteroides Gonadales/análisis , Hormonas Esteroides Gonadales/sangre , Masculino , Embarazo , Valores de ReferenciaRESUMEN
Proteomic analysis of bovine conceptus fluid proteins during early pregnancy has the potential to expose protein species indicative of both the overall health of the fetal-maternal environment and fetal developmental status. In this study, we examined the differential abundance of bovine conceptus fluid proteins (5-50 kDa fraction) from naturally conceived, in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT)-derived pregnancies at days 45 and 90 of gestation. In day 45 allantoic fluid (AllF) samples, an atypical cluster of low molecular weight ( approximately 14-16 kDa), low pI (between 3.0 and 4.5 pH units) protein species was increased in three of four IVF samples (30-100-fold increase in protein spot volumes compared to normal). These proteins were identified as paralogs of the bovine cathelicidin antimicrobial protein (CAMP) by MALDI-TOF MS peptide mass fingerprint and MALDI-TOF MS/MS peptide sequence analysis. Peptidoglycan recognition protein and serine (or cysteine) proteinase inhibitor clade B1, were also significantly increased in the corresponding IVF samples. In two of four SCNT AllF samples, a 2-10-fold increase in CAMP protein spot volumes were detected. No aberrant abundance levels of individual protein species were observed in amniotic fluid samples, or in day 90 IVF AllF samples. Identification of unique protein species present in the normal bovine AllF proteome at day 45 is also reported.
Asunto(s)
Líquido Amniótico/metabolismo , Proteoma/metabolismo , Proteómica , Técnicas Reproductivas Asistidas , Alantoides/química , Alantoides/metabolismo , Secuencia de Aminoácidos , Líquido Amniótico/química , Animales , Péptidos Catiónicos Antimicrobianos/análisis , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Catelicidinas , Bovinos , Electroforesis en Gel Bidimensional , Femenino , Datos de Secuencia Molecular , Técnicas de Transferencia Nuclear , Embarazo , Proteoma/análisis , Inhibidores de Serina Proteinasa/análisis , Inhibidores de Serina Proteinasa/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A proteomic analysis of bovine amniotic and allantoic fluids collected around Day 45 of gestation was performed using gel-based and LC-based MS workflows. A depletion/enrichment protocol using ultrafiltration under denaturing and reducing conditions produced an enriched fraction containing protein species predominantly between 5 and 50 kDa molecular weight. The analyses of conceptus fluid proteins were performed using two strategies; first, 2-DE coupled with MALDI-TOF-MS/MS and LC-ESI-MS/MS analysis of individual protein spots and second, a global protein snapshot of the enriched 5-50 kDa protein fraction by LC-ESI-MS/MS and LC-MALDI-TOF-MS/MS. Allocation of bovine specific protein identities was achieved by searching the Interactive Bovine In Silico SNP (IBISS) and NCBInr protein sequence databases resulting in the confident PMF identification and MS/MS confirmation of >200 2-DE generated allantoic fluids protein spots (74 individual protein species identified) and the MS/MS peptide identification of 105 LC-ESI-MS/MS generated protein identities. In total, the identity of 139 individual protein species from allantoic fluids was confirmed with peptide sequence probability MOWSE scores at the p<0.05 level or better. The comparison of bovine Day 45 amniotic and allantoic fluids protein profiles revealed differences between these two conceptus fluids in early pregnancy.
Asunto(s)
Alantoides/química , Líquido Amniótico/química , Proteínas Gestacionales/química , Proteómica , Alantoides/metabolismo , Líquido Amniótico/metabolismo , Animales , Bovinos , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Femenino , Intercambio Materno-Fetal/fisiología , Embarazo , Proteínas Gestacionales/metabolismo , Análisis por Matrices de Proteínas , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
A capillary zone electrophoresis method was developed for fingerprint determination of allantoic fluid in specific pathogen free (SPF) embryonated chicken eggs. The effects of some crucial parameters, such as buffer type, pH, wavelength and running voltage on the separation were studied systematically. The components of the allantoic fluid were well separated using a fused-silica uncoated capillary with an effective length of 50 cm and an internal diameter of 50 microm. One hundred millimolars sodium tetraborate buffer containing 20 mM sodium dihydrogen phosphate with a final pH 9.8 was used as a running buffer. Comparative fingerprints of allantoic fluid in normal and infected with infectious bronchitis virus (IBV) SPF embryonated chicken eggs were also evaluated. The results showed that there were significant differences between composition of normal allantoic fluid and allantoic fluid infected with IBV, which led to different migration behavior. This method was shown to be stable and reproducible with a relative standard deviation of less than 5% for both migration time and peak current.
Asunto(s)
Alantoides/química , Líquido Amniótico/química , Electroforesis Capilar/métodos , Animales , Embrión de Pollo/virología , Concentración de Iones de Hidrógeno , Virus de la Bronquitis Infecciosa , Reproducibilidad de los Resultados , Organismos Libres de Patógenos EspecíficosRESUMEN
Single-domain antibodies are attractive as tumor-targeting vehicles because of their much smaller size than intact antibody molecules. Lidamycin is a macromolecular antitumor antibiotic, which consists of a labile enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). An enediyne-energized fusion protein VH-LDP-AE composed of single-domain antibody directed against type IV collagenase and lidamycin was prepared by a novel two-step method including DNA recombination and molecular reconstitution. VH-LDP-AE demonstrated extremely potent cytotoxicity to cancer cells and marked antiangiogenic activity in vitro. In the mouse hepatoma 22 model, drugs were administered intravenously as a single dose on day 1 with maximal tolerated doses. VH-LDP-AE (0.25 mg/kg) suppressed the tumor growth by 95.9%, whereas lidamycin (0.05 mg/kg) and mitomycin (1 mg/kg) by 79.6 and 51.1%, respectively. In the HT-1080 xenograft model in nude mice, drugs were given intravenously as a single dose on day 4 after tumor implantation. VH-LDP-AE at 0.25 mg/kg suppressed tumor growth by 76% (P<0.05) compared with that of lidamycin at 0.05 mg/kg (53%) on day 18. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein VH-LDP-AE was more effective than lidamycin and mitomycin. These properties, together with its much smaller size than conventional antibody-based agents, suggested that VH-LDP-AE would be a promising candidate for cancer-targeting therapy. In addition, the two-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.
Asunto(s)
Anticuerpos/química , Anticuerpos/farmacología , Antineoplásicos/química , Antineoplásicos/farmacología , Alantoides/química , Alantoides/efectos de los fármacos , Secuencia de Aminoácidos , Inhibidores de la Angiogénesis/síntesis química , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/síntesis química , Western Blotting , Línea Celular Tumoral , Embrión de Pollo , Colagenasas/química , Colagenasas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Vectores Genéticos , Indicadores y Reactivos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente , Microtúbulos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
REASONS FOR PERFORMING STUDY: Most current treatments for placentitis in mares are empirical with few control studies to evaluate their effectiveness. OBJECTIVE: To monitor drug concentrations in allantoic fluid of pregnant pony mares using in vivo microdialysis and establish if this method would be useful for determining allantoic concentrations of drugs in normal mares and those with placentitis. METHODS: Five late gestational pony mares had microdialysis probes inserted into the allantoic fluid using transabdominal ultrasound-guided allantocentesis. Single injections of penicillin G (22,000 u/kg), gentamicin (6.6 mg/kg bwt) and flunixin meglumine (1 mg/kg bwt) were administered i.v. and dialysate samples collected continuously for 24 h. In a separate study, drug concentrations were monitored in allantoic fluid of 2 mares with experimental placentitis induced by intracervical inoculation with Streptococcus equi ssp. zooepidemicus. Drug concentrations were measured by high performance liquid chromatography (penicillin G, flunixin meglumine) or enzyme-linked immunosorbent assay (gentamicin). RESULTS: Penicillin G and gentamicin achieved average peak concentrations of 9.8+/-2.2 and 8.5+/-3.1 microg/ml, respectively, in allantoic fluid of noninfected mares. Pharmacokinetic comparisons indicate that penicillin G persists much longer in allantoic fluid than blood, whereas gentamicin exhibited similar profiles in the 2 compartments. Flunixin meglumine was not detected in allantoic fluid. In infected mares, penicillin G achieved a similar peak concentration in allantoic fluid (11.2 microg/ml) whereas peak gentamicin concentration (3.9 microg/ml) appeared to be reduced relative to drug concentrations in noninfected mares. CONCLUSIONS: Microdialysis is a useful technique for continuous in vivo monitoring of drugs in equine allantoic fluid. Our results indicate that penicillin G and gentamicin undergo effective placental transfer in pregnant mares and in 2 mares that transplacental drug transfer may be altered selectively if active placental infection is present. POTENTIAL RELEVANCE: Further studies are needed to evaluate the feasibility of using increased dose intervals for penicillin G and an increased dose rate of gentamicin to effectively combat placental infections in mares.
Asunto(s)
Líquido Amniótico/metabolismo , Antibacterianos/farmacocinética , Gentamicinas/farmacocinética , Enfermedades de los Caballos/metabolismo , Microdiálisis/veterinaria , Penicilina G/farmacocinética , Placenta/metabolismo , Alantoides/química , Alantoides/metabolismo , Líquido Amniótico/química , Animales , Antibacterianos/análisis , Área Bajo la Curva , Femenino , Gentamicinas/análisis , Caballos , Tasa de Depuración Metabólica , Microdiálisis/métodos , Penicilina G/análisis , EmbarazoRESUMEN
In the literature, IGFs in the developing embryo are usually determined by blood serum concentrations. For this study, IGF-I/-II was quantified in the amniotic and allantoic fluids of fertile commercial broiler chicken (Gallus domesticus) (n=222), Pekin duck (Anas platyrhyncha) (n=250), and turkey (Meleagridis gallopavo) eggs (n= 200) during incubation. Amniotic and allantoic fluids were collected from embryos starting at 6 days of incubation for chickens and 8 days of incubation for ducks and turkeys. IGF concentrations within the fluids were determined by radioimmunoassay. Chicken amniotic IGF-I concentration at stage 29 of development was significantly higher (P< or =0.05) than the duck or turkey. At stage 36 of development the concentration of IGF-II in the amniotic fluid was 2.8 times greater in the chicken versus the duck (P< or =0.05) and 2 times greater than in the turkey (P< or =0.05). Within species, chicken IGF-I concentration in the amniotic fluid had a cubic trend (P< or =0.001), duck IGF-I increased linearly (P< or =0.001), and turkey concentrations declined quadratically (P< or =0.001) throughout development. In all species, the IGF-II concentration was higher than the IGF-I concentration in the amniotic and allantoic fluids.
Asunto(s)
Alantoides/metabolismo , Líquido Amniótico/metabolismo , Patos/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Pavos/metabolismo , Alantoides/química , Líquido Amniótico/química , Animales , Embrión de Pollo , Patos/embriología , Embrión no Mamífero , Factor I del Crecimiento Similar a la Insulina/análisis , Factor II del Crecimiento Similar a la Insulina/análisis , Radioinmunoensayo , Especificidad de la Especie , Pavos/embriologíaRESUMEN
Recent studies examining patterns and consequences of variation in maternally deposited steroids in avian egg yolk have demonstrated that these maternal hormones can have dramatic effects on chick phenotypes. However, maternal steroids are not the only source for avian embryos, which activate endocrine axes relatively early in development and are capable of producing substantial amounts of endogenous steroids. Although organizational effects of steroids have been demonstrated, the interactions between steroids from yolk and endogenous production have not been addressed. Steroids in the yolk are likely to alter development of the embryo's endocrine axes. The ability to assess total steroid exposure in ovo in a nonlethal fashion would improve our understanding of these interactions and help elucidate the mechanisms by which maternal steroids alter chick phenotype. Steroid levels in allantoic waste provide a cumulative measure of steroids excreted in ovo and may prove to be a useful tool. We present data from semiprecocial seabirds, common murres, demonstrating the presence of detectable steroids in allantoic waste and suggesting that some reflect differences in timing of hatching and may provide information about aspects of chick phenotype.
Asunto(s)
Alantoides/química , Aves/metabolismo , Óvulo/química , Esteroides/análisis , Animales , Femenino , MasculinoRESUMEN
To characterize the maternal-fetal transport of lipophilic endocrine disrupting chemicals, concentrations of polychlorinated (2,3,7,8-substituted) dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs) were measured in maternal and fetal blood, and amniotic and allantoic fluids in cattle. Total toxicity equivalent quantity (TEQ) was highest in amniotic fluid on a fat-weight basis, whereas it was highest in maternal blood on a total weight basis. TEQ was lowest in allantoic fluid on either basis; 26 of 29 congeners analyzed in this experiment were detected in one or more samples. The largest number of congeners was detected in amniotic fluid. O8CDD, 2,3,4,7,8-P5CDF and 2,3',4,4',5-P5CB were the major congeners in PCDDs, PCDFs and PCBs, respectively. The O8CDD concentration was higher in fetal blood than in maternal blood on a fat-weight basis, whereas concentrations of other congeners were lower in fetal blood than in maternal blood. Furthermore, on a fat-weight basis, the O8CDD concentration was considerably higher in allantoic fluid compared with other samples. Concentrations of major PCB congeners were higher in amniotic fluid than in maternal and fetal blood on a fat-weight basis. In conclusion, it is suggested that lipophilic endocrine-disrupting chemicals contained in maternal blood are all transferred to the fetal circulation via the placenta in cattle. Furthermore, the results of this experiment imply that O8CDD has different transportation systems from other dioxins in the circulation, and that a considerable amount of PCBs is excreted and accumulated in amniotic fluid during the fetal stage in cattle.
Asunto(s)
Alantoides/química , Líquido Amniótico/química , Benzofuranos/sangre , Sangre Fetal/química , Bifenilos Policlorados/sangre , Dibenzodioxinas Policloradas/análogos & derivados , Dibenzodioxinas Policloradas/sangre , Animales , Bovinos , Femenino , EmbarazoRESUMEN
We investigated the feasibility of using Flinders Technology Associates (FTA) filter cards for the storage of allantoic fluid containing an infectious bronchitis virus (IBV), such as Arkansas-DPI, Connecticut, and Massachusetts, and for their identification by reverse transcriptase (RT)-polymerase chain reaction (PCR) and characterization by restriction fragment length polymorphism (RFLP) or nucleotide sequencing. FTA paper is a cotton-based cellulose membrane containing lyophilized chemicals that lyses many types of bacteria and viruses. IBV was inactivated upon contact with the FTA, as shown by the inability of the virus to be propagated in embryonating chicken eggs. RT-PCR of the S1 gene showed that viral RNA in allantoic fluid remained stable after storage on FTA filter cards and that the stability was time and temperature sensitive for the large (1700 base pair [bp]) but not the small (383 bp) PCR products. Analysis of the amplified products showed that molecular characterization is feasible in allantoic fluid stored on FTA under nonfavorable environmental conditions (41 C) for at least 15 days. The use of FTA cards for the collection, transport, and storage of IBV-containing samples is safe, inexpensive, and adequate for molecular diagnosis. We propose that specimens coming from overseas on FTA cards would be first analyzed by RT-PCR with primers yielding a 1700-bp product followed by RFLP of the positive cases. Negative cases would be analyzed with primers yielding a 383-bp product (to exdude detrimental effect of the storage conditions) followed by nucleotide sequencing of the positive cases.
Asunto(s)
Pollos/virología , Líquido Extracelular/virología , Virus de la Bronquitis Infecciosa/genética , Manejo de Especímenes/veterinaria , Alantoides/química , Animales , Biología Computacional , Cartilla de ADN , Filtración/instrumentación , Papel , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/veterinaria , Manejo de Especímenes/instrumentación , Temperatura , Factores de Tiempo , Inactivación de VirusRESUMEN
Extracellular magnesium salts are known to interfere with ionic channels in the cellular membranes. The membrane potential, a regulator of vascular tone, is a function of the physiological activities of ionic channels (particularly, K+ and Ca2+ channels in these cells). These channels regulate the ionic distribution into these cells. Micro-Particule Induced X-ray Emission (PIXE) analysis was applied to determine the ionic composition of vascular smooth muscle cells (VSMC) and of vascular endothelial cells (VEC) in the placental human allantochorial vessels in a physiological medium (Hanks' solution) modified by the addition of 2 mM MgCl2 or 2 mM MgSO4 which block the calcium-sensitive K+ channels (K(Ca)), the ATP-sensitive K+ channels (K(ATP)) and the voltage-sensitive K+ (K(df)) and Ca2+ channels. In VSMC (media layer), the addition of MgCl2 induced no modification of the K, Cl, P, S and Ca concentrations but increased the Na and Mg concentrations and the addition of MgSO4 only significantly increased the Mg concentration, the other ion concentrations remaining constant. In endothelium (VEC), MgCl2 or MgSO4 addition implicated the same observations as in VSMC. These results confirmed the blockage of K(df), K(Ca), K(ATP) and Ca channels in VSMC and VEC by magnesium salts, the relationship between Mg2+ ions and internal Na and demonstrated the possible intervention of a Na+/Mg2+ exchanger.
Asunto(s)
Alantoides/irrigación sanguínea , Corion/irrigación sanguínea , Elementos Químicos , Cloruro de Magnesio/farmacología , Sulfato de Magnesio/farmacología , Placenta/irrigación sanguínea , Alantoides/química , Corion/química , Endotelio Vascular/química , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Femenino , Humanos , Músculo Liso Vascular/química , Músculo Liso Vascular/efectos de los fármacos , Placenta/química , Placenta/metabolismo , EmbarazoRESUMEN
In this study we investigated the effects of glycine on angiogenesis during embryogenesis, wound healing and tumor growth. In chorioallantoic membrane (CAM) assay, glycine (100 microM) inhibited angiogenesis by more than 50%. We studied dietary glycine's effect on fibrin induced wound healing response in a novel (Fibrin Z-chamber) assay. Fibrin within the chamber triggers the healing cascade leading to formation of granulation tissue (GT) rich in blood vessels and stroma. GT was reduced by more than 30% (p < 0.0001) in dietary Glycine groups as compared to control. We found that microvessel density dropped significantly (15%, p < 0.0003) with dietary glycine whereas the other components of GT were unaffected. We evaluated tumor growth delay utilizing Tumor Z-Chamber (fibrin with R3230 mammary adenocarcinoma cells) since tumors take advantage of angiogenesis and matrix formation. We observed that tumor growth decreased by 15% (p < 0.03) and tumor microvessel density dropped by 20% (p < 0.03) with dietary glycine compared to controls. We found that iNOS protein levels were decreased significantly in both GT (24%-57%) and tumor tissue (19-75%). In conclusion, we found that dietary glycine is a potent anti-angiogenic agent that can reduce wound healing and tumor growth through reduction of iNOS expression.
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Inhibidores de la Angiogénesis/administración & dosificación , Glicina/administración & dosificación , Neoplasias Mamarias Experimentales/prevención & control , Neovascularización Patológica/prevención & control , Cicatrización de Heridas/efectos de los fármacos , Alantoides/química , Animales , Western Blotting , División Celular , Corion/química , Corion/metabolismo , Dieta , Femenino , Fibrina/metabolismo , Geles , Tejido de Granulación/enzimología , Tejido de Granulación/metabolismo , Técnicas para Inmunoenzimas , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Neovascularización Patológica/enzimología , Neovascularización Patológica/patología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas Endogámicas F344RESUMEN
David Peakall and co-workers pioneered innovative approaches that utilized extra-embryonic membranes to assess accumulation of organochlorine pesticides in eggs. This technique provided the foundation for an entire line of research to improve non-lethal methods for assessing contaminant exposure in oviparous wildlife. Currently, analysis of chorioallantoic membranes (CAMs) provides predictable estimates of chlorinated contaminant presence in eggs and in maternal tissues. Field studies have been conducted with herons, stilts, alligators, crocodiles, and sea turtles. Controlled dose-response studies have been completed in chickens. The following manuscript presents the foundations for the CAM approach and a review of research findings involving this technique.
Asunto(s)
Alantoides/química , Animales Salvajes , Corion/química , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/farmacocinética , Animales , Aves , Predicción , Compuestos Orgánicos/farmacocinética , Reptiles , Distribución TisularRESUMEN
Serum prolactin increases during late embryogenesis. In order to elucidate the function of prolactin at this period, tissue distribution of prolactin receptor mRNA was examined by RNase protection assay. The mRNA was detected strongly in the kidney, intestine, and allantoic membrane; weakly detected in the brain; but not detected in the liver. The expression levels of the prolactin receptor mRNA in the kidney, intestine, and allantoic membrane were retained at constant levels during later stages of embryogenesis (Days 17 and 19) and posthatch periods (2 and 28 d after hatching). These results suggest that prolactin is mainly involved in the osmoregulation during the later stage of embryogenesis and that the expression of prolactin receptor mRNA in the kidney, intestine, and allantoic membrane is regulated by a serum prolactin-independent manner.
Asunto(s)
Embrión de Pollo/química , Embrión de Pollo/crecimiento & desarrollo , ARN Mensajero/análisis , Receptores de Prolactina/genética , Alantoides/química , Alantoides/embriología , Animales , Encéfalo/embriología , Química Encefálica , Intestinos/química , Intestinos/embriología , Riñón/química , Riñón/embriología , Factores de Tiempo , Distribución TisularRESUMEN
To gain insight into the role of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzymes and actions of glucocorticoids in the murine placenta and uterus, the expression pattern of the mRNA for 11beta-HSD1 and 11beta-HSD2 and the glucocorticoid receptor (GR) protein were determined from Embryonic Day 12.5 (E12.5, term = E19) to E18.5 by in situ hybridization and immunohistochemistry, respectively. Consistent with its putative role in regulating the transplacental passage of maternal glucocorticoid to the fetus, 11beta-HSD2 mRNA was highly expressed in the labyrinthine zone (the major site of maternal/fetal exchange) at E12.5, and its level decreased dramatically at E16.5, when it became barely detectable. Remarkably, the silencing of 11beta-HSD2 gene expression coincided with the onset of 11beta-HSD1 gene expression in the labyrinth at E16.5 when moderate levels of 11beta-HSD1 mRNA were detected and maintained to E18.5. By contrast, neither 11beta-HSD1 mRNA nor 11beta-HSD2 mRNA were detected in any cell types within the basal zone from E12.5 to E18.5. Moreover, the expression of 11beta-HSD1 and 11beta-HSD2 in the decidua exhibited a high degree of cell specificity in that the mRNA for both 11beta-HSD1 and 11beta-HSD2 was detected in the decidua-stroma but not in the compact decidua. A distinct pattern was also observed within the endometrium where the mRNA for 11beta-HSD1 was expressed in the epithelium, whereas that for 11beta-HSD2 was confined strictly to the stroma. By comparison, the expression of GR in the placenta and uterus was ubiquitous and unremarkable throughout late pregnancy. In conclusion, the present study demonstrates for the first time remarkable spatial and temporal patterns of expression of 11beta-HSD1 and 11beta-HSD2 and GR in the murine placenta and uterus and highlights the intricate control of not only transplacental passage of maternal glucocorticoid to the fetus but also local glucocorticoid action during late pregnancy.
Asunto(s)
Hidroxiesteroide Deshidrogenasas/genética , Placenta/química , ARN Mensajero/análisis , Receptores de Glucocorticoides/análisis , Útero/química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2 , Alantoides/química , Animales , Corion/química , Decidua/química , Endometrio/química , Epitelio/química , Femenino , Edad Gestacional , Inmunohistoquímica , Hibridación in Situ , Ratones , Miometrio/química , Embarazo , Células del Estroma/química , Saco Vitelino/químicaRESUMEN
Menstrual cycle activity predisposes to ovarian epithelial tumors based on numerous epidemiological studies. We showed that the hormones involved in menstrual cycle regulation modulate cell cycle activity in these tumors in an accompanying paper. We investigated whether such hormones could also influence angiogenesis, an important determinant of tumor progression, in the same tumors. Treatment with progesterone (P4) stimulated VEGF protein secretion in 4 of 5 ovarian carcinoma cell lines examined. Northern blot analyses performed in MCV50 cells showed that this effect was accompanied by increased VEGF mRNA levels. P4 also stimulated VEGF promoter activity in these cells. Estradiol (E2) showed a similar, but substantially smaller effect on VEGF secretion which was additive to that of P4. Conditioned medium from P4-treated cells strongly stimulated angiogenesis on chicken chorio-allantoic membranes. Incubating the conditioned medium with a neutralizing anti-VEGF antibody, but not with non-specific immunoglobulins abolished this effect. Angiogenic activity was not altered by treatment of the membranes with P4 directly. We conclude that P4 can stimulate angiogenic activity via induction of VEGF secretion in some ovarian epithelial tumors. Therapeutic use of progestins may be most effective when administered in combination with an anti-angiogenic agent, at least against a subset of ovarian carcinomas.
Asunto(s)
Estradiol/uso terapéutico , Neoplasias Glandulares y Epiteliales/irrigación sanguínea , Neovascularización Patológica/tratamiento farmacológico , Neoplasias Ováricas/irrigación sanguínea , Progesterona/uso terapéutico , Alantoides/química , Animales , Northern Blotting , Embrión de Pollo , Corion/química , Factores de Crecimiento Endotelial/genética , Factores de Crecimiento Endotelial/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Luciferasas/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Neovascularización Patológica/metabolismo , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Elementos de Respuesta , Transfección , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The H beta 58 gene, whose disruption in mice causes reabsorption of the embryo at 9.5 days post-conception, is believed to be essential for development of the placenta. Although the H beta 58 gene is well conserved in some Amniota, nothing is known about its presence in reptiles, some species of which have developed a chorioallantoic placenta. In this work, we investigated the expression of H beta 58 mRNA and protein in the three-toed skink, Chalcides chalcides. H beta 58 protein expression was found in the uterine epithelium beginning from the peri-ovulatory stage. However, it increased strongly at the moment of placental formation, when a high level of expression of mRNA and protein was also observed in the extra-embryonic membranes. The expression of H beta 58 mRNA and protein was maintained, although to a lesser degree, in the placenta during late pregnancy. It was also present in the early embryo. Finally, cloning and sequencing of a gene fragment revealed strong homology of the reptile gene with that of mammals. The high degree of conservation of the gene in amniote vertebrates and its presence in a viviparous squamate reptile (as in mammals) indicates an important role of this gene in the chorioallantoic placenta formation and development.