RESUMEN
Introduction: Osteoarthritis (OA) commonly occurs following anterior cruciate ligament reconstruction (ACLR), affecting over 50 % of patients within 10-15 years post-ACLR. The Hoffa-synovitis of the infrapatellar fat pad (IPFP) has been implicated as a major contributor to OA pathogenesis. While MRI is typically used to evaluate the IPFP, it is cost-prohibitive for routine screening. This study aimed to validate ultrasound as an alternative for detecting IPFP Hoffa-synovitis in participants post-ACLR. Methods: In this cross-sectional study, 15 participants (18-35 years, 1-5 years post-ACLR) underwent two imaging sessions separated by one week. First, a standardized bilateral anterior knee ultrasound assessment was used to examine IPFP echo-intensity. Second, MRI scans of both knees were graded by a board-certified musculoskeletal radiologist for Hoffa-synovitis according to the Anterior Cruciate Ligament Osteoarthritis Score grading system. IPFP echo-intensity were quantified on each ultrasound image, and a limb symmetry index (LSI) was calculated to assess between-limb differences. We used an independent t-test and Cohen's d effect sizes to compare IPFP echo-intensity LSI between people with and without MRI-confirmed Hoffa-synovitis. Results: Four of the 15 participants (27 %) exhibited MRI-confirmed Hoffa-synovitis. Significantly higher IPFP echo-intensity LSI values were found in participants with Hoffa-synovitis (32.1 ± 12.1 %) compared to those without (10.5 ± 10.4 %), confirming the ultrasound's ability to distinguish between the two groups (t = -3.44; p = 0.004; d = 2.01). Discussion: Ultrasound detects bilateral IPFP signal intensity alterations in participants post-ACLR with MRI-confirmed Hoffa-synovitis. This work should be seen as a proof-of-concept, and further validation in a larger, more diverse sample is essential for verifying these results.
RESUMEN
Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.
Asunto(s)
Alelos , Perros/genética , Antígenos de Histocompatibilidad Clase I/genética , Polimorfismo Genético , Animales , Perros/inmunología , Técnicas de Genotipaje , Antígenos de Histocompatibilidad Clase I/inmunologíaRESUMEN
OBJECTIVE: To determine the magnitude of medial femoral cartilage deformation using ultrasonography (US) following walking and running in healthy individuals. DESIGN: Twenty-five healthy participants with no history of osteoarthritis or knee injury volunteered for this study. Medial femoral cartilage thickness was assessed using US before and after three separate 30-min loading conditions: (1) walking at a self-selected speed, (2) running at a self-selected speed, and (3) sitting on a treatment table (i.e., control). Cartilage deformation was calculated as the percent change score from pre to post loading in each loading condition. The magnitude of cartilage deformation was compared between the three loading conditions. RESULTS: There was no difference in baseline cartilage thickness between the three sessions (F1,24 = 0.18, P = 0.68). Cartilage deformation was different between the loading conditions (F1,24 = 47.54, P < 0.001). The walking (%Δ = -6.7, t24 = 6.90, P < 0.001, d = -1.92) and running (%Δ = -8.9, t24 = 8.14, P < 0.001, d = -1.85) conditions resulted in greater cartilage deformation when compared to the control condition (%Δ = +3.4). There was no difference in cartilage deformation between the running and walking conditions (t24 = 1.10, P = 0.28, d = 0.33). US measured medial femoral cartilage thickness demonstrated reliability and precision within a single session (ICC2,k = 0.966, SEM = 0.07 mm) and between additional sessions separated by seven (ICC2,k = 0.964, SEM = 0.08 mm) and 16 days (ICC2,k = 0.919, SEM = 0.11 mm). CONCLUSIONS: US demonstrated to be a reliable and sensitive imaging modality at quantifying medial femoral cartilage deformation in healthy individuals. Both walking and running conditions created greater cartilage deformation when compared to the control conditions, but no difference was observed between the walking and running conditions.
Asunto(s)
Cartílago Articular/diagnóstico por imagen , Articulación de la Rodilla/diagnóstico por imagen , Carrera/fisiología , Caminata/fisiología , Soporte de Peso/fisiología , Cartílago Articular/patología , Cartílago Articular/fisiología , Femenino , Voluntarios Sanos , Humanos , Articulación de la Rodilla/fisiología , Masculino , Tamaño de los Órganos , Ultrasonografía , Adulto JovenRESUMEN
OBJECTIVE: There is an increased risk of developing knee osteoarthritis (OA) following anterior cruciate ligament (ACL) injury. Biomarkers may provide diagnostic, prognostic, or burden of disease indicators of OA before radiographic changes become apparent. Unfortunately, there has been no systematic review to clarify which biomarkers may be most informative following injury. Therefore, this review critically investigated existing studies of OA-related biomarkers in ACL-deficient (ACL-D) and reconstructed (ACL-R) patients to summarize the current evidence and identify knowledge gaps. DESIGN: A systematic review of the literature in Web of Science and PubMed databases (1960-June 2014) was performed. All English-language case-control and longitudinal studies assessing OA-related biomarkers in ACL-D and ACL-R patients were considered. Data regarding biomarker changes over time within ACL-D and ACL-R patients as well as differences in ACL-D/ACL-R patients compared with a control group were extracted from pertinent studies. RESULTS: A descriptive summary of 20 included studies was produced. In ACL-D patients compared with controls, synovial fluid biomarkers indicated elevated collagen turnover, while the inflammatory cytokine response was inconclusive. In ACL-R patients, serum concentrations indicated decreased collagen breakdown, but urine concentrations were indicative of greater collagen breakdown when compared to controls. Compared to preoperative values, the overall inflammatory cytokine response measured with synovial fluid biomarkers increased while plasma biomarkers did not change following reconstruction. CONCLUSION: Patients with ACL-D or ACL-R have altered biomarkers indicative of OA. More research with standardized reporting is needed to effectively determine which biomarkers are the most indicative for OA development and progression following ACL injury.
Asunto(s)
Lesiones del Ligamento Cruzado Anterior , Reconstrucción del Ligamento Cruzado Anterior , Ligamento Cruzado Anterior/cirugía , Osteoartritis de la Rodilla/diagnóstico , Complicaciones Posoperatorias/diagnóstico , Biomarcadores/análisis , Estudios de Casos y Controles , HumanosRESUMEN
The effects of the herbal product kava (Kava kava, 'Awa, Yaqona, Piper methysticum) on human P450 isoforms were studied in vitro using both cDNA-expressed human enzymes and cryopreserved human hepatocytes. Increasing concentrations of an ethanolic extract of dried kava root and three purified kava lactones (methysticin, desmethoxyyangonin, and yangonin) were tested for their ability to inhibit the catalytic activity of a panel of P450 isoforms (1A2, 2A6, 2C9, C2C19, 2D6, 2E1, and 3A4) present as c-DNA expressed-enzymes and in previously cryopreserved human hepatocytes. In addition, the test compounds' effect on hepatocyte viability was evaluated by measuring cellular ATP content. In both models, the kava extract and the three kava lactones were found to be potent inhibitors of CYPs 1A2, 2C9, 2C19, 2E1, and 3A4 with IC50 values of approximately 10 microM. The test compounds were also moderately cytotoxic to human hepatocytes (EC50 values of approximately 50 microM). Methysticin was the most potent enzyme inhibitor as well as the most cytotoxic, followed by (in order of potency:) the kava root extract, desmethoxyyangonin, and yangonin. Our results suggest that the drug interaction and hepatotoxic potential of kava should be further investigated.
Asunto(s)
Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hepatocitos/efectos de los fármacos , Kava , Fitoterapia , Extractos Vegetales/farmacología , Criopreservación , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/efectos de los fármacos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/uso terapéutico , Hepatocitos/enzimología , Humanos , Concentración 50 Inhibidora , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Raíces de Plantas , Piranos/farmacología , Pironas/farmacologíaRESUMEN
We evaluated the effects of 25 purified components of commonly used herbal products on the catalytic activity of cDNA-expressed cytochrome P450 isoforms in in vitro experiments. Increasing concentrations of the compounds were incubated with a panel of recombinant human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. For each test substance, the IC50 (the concentration required to inhibit metabolism of surrogate substrates by 50%) was estimated and compared with IC50's for the positive control inhibitory drugs furafylline, sulfaphenazole, tranylcypromine, quinidine, and ketoconazole. Constituents of Ginkgo biloba (ginkgolic acids I and II), kava (desmethoxyyangonin, dihydromethysticin, and methysticin), garlic (allicin), evening primrose oil (cis-linoleic acid), and St. John's wort (hyperforin and quercetin) significantly inhibited one or more of the cDNA human P450 isoforms at concentrations of less than 10 uM. Some of the test compounds (components of Ginkgo biloba, kava, and St. John's wort) were more potent inhibitors of the isoforms 1A2, 2C19, and 2C19 than the positive controls used in each assay (furafylline, sulfaphenazole, and tranylcypromine, respectively), which are known to produce clinically significant drug interactions. The enzyme most sensitive to the inhibitory of effects of these compounds was CYP2C19, while the isoform least effected was CYP2D6. These data suggest that herbal products containing evening primrose oil, Ginkgo biloba, kava, and St. John's Wort could potentially inhibit the metabolism of co-administered medications whose primary route of elimination is via cytochrome P450.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/biosíntesis , Inhibidores Enzimáticos/farmacología , Extractos Vegetales/farmacología , Biomarcadores , Catálisis/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450 , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
To evaluate the effects of intrinsic (natural) fluorescence and quenching as confounding variables in fluorescence-based enzyme inhibition assays of natural products, we measured the fluorescence and quenching properties of 25 components of popular herbal products. The analyses were performed under conditions typically employed in drug-drug interaction studies that use c-DNA-derived P450 isoforms and surrogate fluorogenic substrates. Four of the 25 compounds tested (isorhamnetin, quercetin, vitexin, and yangonin) fluoresced or quenched sufficiently to interfere with these assays. Intrinsic fluorescence had a greater effect on these assays than quenching and for one compound, yangonin, was sufficient to mask inhibition and potentially produce a false negative result. Quenching had less of an effect on these assays, but was significant enough for one compound, quercetin, to mimic "weak" inhibition. Therefore, because intrinsic fluorescence or quenching could render some natural products unsuitable for testing in certain fluorometric assays, it would be prudent to include an evaluation of these properties in experimental protocols.
Asunto(s)
Apigenina , Enzimas/efectos de los fármacos , Flavonoles , Extractos Vegetales/farmacología , Hidrocarburo de Aril Hidroxilasas/efectos de los fármacos , Citocromo P-450 CYP1A2/efectos de los fármacos , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6/efectos de los fármacos , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Flavonoides/farmacología , Fluorescencia , Humanos , Oxigenasas de Función Mixta/efectos de los fármacos , Fitoterapia , Extractos Vegetales/química , Pironas/farmacología , Quercetina/análogos & derivados , Quercetina/farmacologíaRESUMEN
An HPLC-MS/MS method was developed for the quantitative determination of ginsenosides, which are the marker compounds for herbal products containing Panax ginseng (Korean or Chinese ginseng) and P. quinquefolius (American ginseng). Samples were extracted with BondElut C18 HF extraction columns and the concentrations of seven major ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) were determined by reversed-phase HPLC-MS/MS employing a quadrupole-ion trap mass spectrometer. Both positive and negative electrospray ionisation techniques were evaluated. Positive ionisation spectra of these compounds gave strong sodium adduct molecular and sodium adduct dimer ions. Negative ionisation yielded the molecular ion primarily and was, therefore, used for analysis: quantitative determination was based on the most abundant product ions for each ginsenoside. The method was used to extract and analyse commercial samples of P. ginseng and P. quinquefolius.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Panax/química , Saponinas/análisis , GinsenósidosRESUMEN
Oncoretroviral vectors require division of target cells for successful transduction. In the case of hematopoietic repopulating cells this can be achieved by cytokine stimulation using growth factor combinations which facilitate gene transfer and maintain engraftment. Interleukin-3 (IL-3) has been widely used in growth factor combinations, although more recent data in the mouse showed reduced engraftment in the presence of IL-3. Here, we used a competitive repopulation assay to study the influence of IL-3 and the early acting cytokines megakaryocyte growth and development factor (MGDF) and Flt3-ligand (Flt3-L) on gene transfer efficiency during ex vivo transduction of hematopoietic repopulating cells. In a direct comparison, baboon CD34-enriched cells were transduced on CH-296 fibronectin fragment in the presence of either IL-6, stem cell factor (SCF), Flt3-L, and MGDF or IL-3, IL-6, and SCF. Animals were followed for up to 55 weeks, and analysis of peripheral blood leukocytes by semiquantitative polymerase chain reaction showed that both cytokine combinations achieved marking of repopulating cells. A trend toward increased gene marking, especially early after transplant (P = 0.06), was seen with the combination of IL-6, SCF, Flt3-L, and MGDF. However, the highest gene marking was achieved when IL-3 was combined with early acting cytokines, suggesting that the difference observed in this study was probably due to the addition of MGDF and Flt3-L and not due to a negative effect of IL-3 on engraftment.
Asunto(s)
Trasplante de Médula Ósea , Fibronectinas/genética , Técnicas de Transferencia de Gen , Interleucina-3/farmacología , Proteínas de la Membrana/farmacología , Protectores contra Radiación/farmacología , Proteínas Recombinantes/genética , Retroviridae/genética , Trombopoyetina/farmacología , Animales , Antígenos CD34/metabolismo , Southern Blotting , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Técnicas de Cocultivo , Ensayo de Unidades Formadoras de Colonias , Cartilla de ADN/química , Citometría de Flujo , Terapia Genética/métodos , Vectores Genéticos , Virus Helper/química , Humanos , Interleucina-6/farmacología , Papio , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/farmacología , Factor de Células Madre/farmacología , TransfecciónRESUMEN
BACKGROUND: Because dietary supplements are not subject to the same regulations that pharmaceuticals are, there is concern among medical professionals that these products may lack purity or potency. OBJECTIVE: To determine the variability in a range of ginseng herbal products available in the United States, we identified and measured the concentration of marker compounds by using HPLC and liquid chromatography-tandem mass spectrometry. DESIGN: Twenty-five commercial ginseng preparations from the genera Panax or Eleutherococcus were obtained from a local health food store and analyzed for 7 ginsenosides (marker compounds for Panax species, which include Asian and American ginseng) and 2 eleutherosides (marker compounds for Eleutherococcus senticosus, also known as Siberian ginseng). RESULTS: All plant products were correctly identified by botanical plant species (ie, Panax species or E. senticosus); however, concentrations of marker compounds differed significantly from labeled amounts. There was also significant product-to-product variability: concentrations of ginsenosides varied by 15- and 36-fold in capsules and liquids, respectively, and concentrations of eleutherosides varied by 43- and 200-fold in capsules and liquids, respectively. Although a systematic search for adulterants was not conducted, review of the HPLC and liquid chromatography-tandem mass spectrometry data suggest that no substances other than ginsenosides or eleutherosides were extracted from the plant material. CONCLUSION: Our data suggest that US ginseng products are correctly labeled as to plant genus; however, variability in concentrations of marker compounds suggests that standardization may be necessary for quality assurance and that characterization of herbal products should be considered in the design and evaluation of studies on herbal products.
Asunto(s)
Suplementos Dietéticos/análisis , Panax , Extractos Vegetales , Plantas Medicinales , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Eleutherococcus , Garantía de la Calidad de Atención de Salud , Estados UnidosRESUMEN
The use of alternative medicine, including consumption of herbal products and dietary supplements, has been increasing substantially both in the United States and in Western Europe. One area that is garnering increased attention is the use of Oriental Medicine including Kampo, or Japanese herbal medicine. Herein, we review representative examples of research available on the most common use of Kampo medicinals, namely to improve the immune response. We also provide an extensive background on the history of Kampo. There are more than 210 different Kampo formulae used in Japan and most uses of Kampo are to modulate the immune response, i.e. to improve immunity. We have extracted data on seven common Kampo medicinals, and the data are reviewed with respect to in vitro and in vivo activities for both humans and experimental animals; the ingredients as well as the problems with classification of these materials are presented. Research suggests that Kampo herbals are biologically active and may have therapeutic potential. While it is believed that Kampo medicines have few side effects, there is a paucity of data on their toxicity as well as a relative lack of knowledge of the bioactive constituents and potential drug interactions of these agents.
Asunto(s)
Antiinflamatorios no Esteroideos/inmunología , Antineoplásicos/inmunología , Medicamentos Herbarios Chinos , Medicina Kampo , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos/uso terapéutico , Medicamentos Herbarios Chinos/uso terapéutico , HumanosRESUMEN
Because little is known about the interactions between herbal products and standard medications, the effects of seven ginsenosides and two eleutherosides (active components of the ginseng root) on the catalytic activity of c-DNA expressed cytochrome P450 isoforms were studied in in vitro experiments. Increasing concentrations of ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 and eleutherosides B and E were incubated with a panel of recombinant human CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) and their effects on the conversion of specific surrogate substrates measured fluorometrically in a 96-well plate format. For each test substance, the IC50 (the concentration required to inhibit the metabolism of the surrogate substrates by 50%) was estimated and this value compared with that obtained for positive control inhibitory drugs furafylline, sulfaphenazole, tryanylcypromine, quinidine, and ketoconizole. Of the components tested, three ginsenosides (Rd, Rc, and Rf) modified the activity of the recombinant enzymes. Ginsenoside Rd produced weak inhibitory activity against the surrogate substrates for CYP3A4 and CYP2D6 and even weaker inhibitory activity against the surrogate substrates for CYP2C19 and CYP2C9. The IC50 values of 58 and 74 uM for the two substrates for CYP3A4 are orders of magnitude higher than that for the potent inhibitor ketoconazole used as a positive control. Ginsenoside Rc produced an increase in the activity of CYP2C9 (70% at 200 uM) and ginsenoside Rf produced an increase in the activity of CYP3A4 (54% at 200 uM). The biological significance of this is unclear at this time. Enzyme "activation", the process by which direct addition of one compound to an enzyme enhances the rate of reaction of the substrate, has been observed in a number of cases with P450 enzymes; however, a matrix effect caused by the test compound fluorescing at the same wavelength as the metabolite of the marker substrate cannot be ruled out. In summary, these studies suggest that the ginsenosides and eleutherosides tested are not likely to inhibit the metabolism of coadministered medications in which the primary route of elimination is via cytochrome P450; the potential of ginsenosides to enhance the catalysis of certain substrates requires further investigation.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/biosíntesis , Inhibidores Enzimáticos/farmacología , Ginsenósidos , Panax/química , Extractos Vegetales/farmacología , Plantas Medicinales , Saponinas/farmacología , Catálisis/efectos de los fármacos , Inhibidores Enzimáticos del Citocromo P-450 , Eleutherococcus , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
In order to evaluate race as a possible factor affecting the incorporation of drugs into human hair, 2 mg/kg deuterium-labeled cocaine (cocaine-d5) was administered intranasally to nine male non-Caucasian volunteers under controlled laboratory conditions. Sequential blood samples were collected for up to three days, and scalp hair samples were collected at 24 and 72 h after dosing and at monthly intervals for up to 12 months. The samples were then analyzed by gas chromatography-mass spectrometry for cocaine-d5 and benzoylegonine-d5 (BZE-d5). The amounts of cocaine-d5 found in the hair of these non-Caucasian subjects were compared with the amounts of cocaine-d5 found in the hair of Caucasian subjects who received the same cocaine dose under identical conditions as part of a study we reported previously. The non-Caucasians in the present study had approximately 2.7 times more cocaine-d5 in their hair than the Caucasian subjects in the earlier study. In five of the non-Caucasian subjects, cocaine-d5 could be detected in hair within 24 h after dosing. Curiously, we were unable to detect any cocaine-d5 in one of the non-Caucasian subject's hair at any time after dosing even though cocaine-d5 was in plasma at the expected levels. The results from these studies suggest there may be a racial bias in the incorporation of cocaine into human hair; however, the data are not conclusive because of the relatively small sample size.
Asunto(s)
Población Negra , Cocaína/farmacocinética , Cabello/metabolismo , Trastornos Relacionados con Sustancias/genética , Población Blanca , Administración Intranasal , Adulto , Población Negra/genética , Cocaína/administración & dosificación , Deuterio , Cromatografía de Gases y Espectrometría de Masas , Humanos , Marcaje Isotópico , Masculino , Detección de Abuso de Sustancias , Trastornos Relacionados con Sustancias/metabolismo , Población Blanca/genéticaRESUMEN
The Swarm rat chondrosarcoma cell line, RCS-LTC, deposits an extracellular matrix that contains the typical type II, IX, and XI collagen phenotype of hyaline cartilage, but the fibrils appear abnormally thin. By N-terminal sequence analysis, the type II collagen from the matrix was shown to have retained its N-propeptides with no evidence of normal processing to type II collagen. Amplification and sequencing of cDNA prepared from the pro alpha1(II) mRNA of these cells showed a normal N-propeptide cleavage site. Furthermore, the type II N-procollagen could be processed to type II collagen by incubation with culture medium from normal chondrocytes. The findings indicate that the RCS-LTC cell line fails to express an active type II procollagen N-proteinase and, therefore, offers a useful culture system in which to study the role of N-propeptide removal in fibrillogenesis.
Asunto(s)
Condrosarcoma/metabolismo , Colágeno/biosíntesis , Procolágeno/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Animales , Colágeno/genética , Colágeno/aislamiento & purificación , ADN Complementario , Exones , Cinética , Datos de Secuencia Molecular , Procolágeno/biosíntesis , Procolágeno/química , Procolágeno N-Endopeptidasa/deficiencia , Procolágeno N-Endopeptidasa/metabolismo , ARN Mensajero/biosíntesis , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Developmental processes associated with skeletogenesis differ in the direct-developing sea urchin Heliocidaris erythrogramma from that in Heliocidaris tuberculata and other indirect-developing species. In H. erythrogramma, the differences include ingression of a much higher number of mesenchyme cells, failure of the cells to form the typical ring pattern of cells prior to the onset of skeletogenesis, a significantly reduced larval skeleton, and a delay in timing of expression of the skeletogenic cell-restricted gene msp130. We report that the heterochronic change in msp130 expression is regulated at the level of transcription. By transient expression of reporter constructs containing msp130 promoter regions from direct- and indirect-developing species, we found that this evolutionary change in regulation is consistent with changes in the timing of action of trans-acting factors in skeletogenic mesenchyme cells. We further used these experiments to show that the H. erythrogramma promoter contains elements required for correct spatial expression in the primary mesenchyme cells of an indirect-developing host. We finally show that alternate processing of H. erythrogramma msp130 is thus far specific to this species and not an aspect of adult skeletogenesis.
Asunto(s)
Evolución Biológica , Regulación del Desarrollo de la Expresión Génica , Erizos de Mar/embriología , Empalme Alternativo , Animales , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Genes Reporteros , Larva , Mesodermo/citología , Mesodermo/fisiología , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Erizos de Mar/genética , Factores de Tiempo , Transcripción GenéticaRESUMEN
To explore the effects of gestational cocaine exposure in a nonhuman primate model, pregnant rhesus monkeys were treated from about 1 month of gestation until term with either 0 (N = 3), 0.3 (N = 3), 1.0 (N = 3), or escalating doses up to 8.5 (N = 3) mg/kg (IM), three times per day, 5 consecutive days per week. Despite these differences in cocaine exposure, the experimental groups did not differ significantly with respect to maternal outcome, as measured by body weight gain during pregnancy and length of pregnancy. A clear dose-response relationship was observed between the cumulative dose of cocaine administered during gestation and the levels of both cocaine and its major metabolite, benzoylecgonine, in samples of infant hair taken at birth. However, the experimental groups did not differ significantly with respect to infant outcome, as measured at birth by body weight, overall length, crown-to-rump length, rump-to-heel length, biparietal diameter, and crown circumference. Furthermore, the experimental groups did not differ significantly with respect to the integrity of a variety of infant reflexes tested at birth. It was concluded that, in a rhesus monkey model, chronic cocaine exposure during pregnancy had no significant effect on maternal and infant outcomes as assessed in this investigation.
Asunto(s)
Cocaína/toxicidad , Narcóticos/toxicidad , Resultado del Embarazo , Efectos Tardíos de la Exposición Prenatal , Animales , Animales Recién Nacidos , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Cocaína/análogos & derivados , Cocaína/análisis , Relación Dosis-Respuesta a Droga , Femenino , Cabello/química , Inyecciones Intramusculares , Macaca mulatta , Narcóticos/análisis , Embarazo , Reflejo/efectos de los fármacosRESUMEN
Deuterium-labeled cocaine (cocaine-d5) was administered intravenously and/or intranasally in doses of 0.6-4.2 mg/kg to 25 human volunteers under laboratory clinical conditions. Sequential blood samples were collected for up to 3 days, and hair samples were collected for up to 10 months. Samples were analyzed by gas chromatography-mass spectrometry (GC-MS) for cocaine-d5 and its major metabolite, benzoylecgonine-d5 (BZE-d5). The parent drug, cocaine-d5, was the predominant analyte in hair, whereas BZE-d5 was the major analyte in blood, especially at later time periods. The amount of cocaine-d5 incorporated into hair ranged from 0.1 to 5 ng/mg hair, whereas the amount of BZE-d5 was approximately one-sixth of that concentration. The threshold dose for detection was estimated to be 25-35 mg of drug administered intravenously. A single dose could be detected for 2-6 months. Subjects receiving the same dose differed (from two to 12 times as much depending upon how it was measured) in the amount of cocaine-d5 incorporated into their hair. Non-Caucasians, in particular, incorporated more cocaine-d5 in hair than did Caucasians. Also, segmental analysis of the samples revealed considerable intersubject variability in the time drug first appeared in hair and the rate at which the drug moved along the hair shaft with time. These interindividual differences could not be explained by differences in plasma pharmacokinetics. Considered together, these results suggest that cocaine incorporation into hair may occur by way of multiple mechanisms--by way of sweat and sebum, for example--and at various times during the hair growth cycle. Thus, hair analysis using GC-MS appears to be a very sensitive method for detecting cocaine ingestion. However, within the range of doses used in the present study, hair does not provide a particularly accurate record of either the amount, time, or duration of drug use.
Asunto(s)
Cocaína/metabolismo , Relación Dosis-Respuesta a Droga , Cabello/metabolismo , Marcaje Isotópico , Administración Intranasal , Adulto , Cocaína/administración & dosificación , Cocaína/sangre , Cocaína/química , Cocaína/farmacocinética , Demografía , Deuterio/química , Cromatografía de Gases y Espectrometría de Masas , Cabello/química , Humanos , Inyecciones Intravenosas , Masculino , Factores de TiempoRESUMEN
Metabolism studies were conducted on 4-methylaminorex (4,5-dihydro-4-methyl-5-phenyl-2-oxazolamine [4-MAX]), a potent central nervous system stimulant that has emerged as a drug of abuse under the name "EU4EA", "EU4Euh", and "Ice". Tritiated norephedrine was cyclized with cyanogen bromide to form 3H-4-MAX, which was administered to rats at a dose of 10 mg/kg orally and intravenously. Radioactivity was excreted almost entirely in urine (40% of the dose was excreted by 24 h), primarily as the parent drug (60% of the total excretions were as the parent compound). Three metabolites were identified by high-performance liquid chromatography-tandem mass spectrometry with thermospray ionization: norephedrine, 5-phenyl-4-methyl-2-oxazolidinone, and 2-amino-5-(p-hydroxyphenyl)-4-methyl-2-oxazoline. Stability studies showed that 4-MAX in aqueous solution degraded very slightly to norephedrine upon standing. There was no evidence for glucuronide or sulfate conjugation. These results suggest that the metabolic fate of 4-MAX is similar to that of the amphetamines in that it is eliminated primarily unchanged but undergoes some slight oxidative deamination and aromatic hydroxylation. Hydrolytic degradation back to the synthetic precursor can also occur. There was no evidence for the hydrolysis of the oxazolamine ring to form a urea that has been reported for the demethylated congener aminorex. This suggests that 4-methyl substitution of the oxazoline ring may inhibit metabolism similar to the alpha-methyl substitution of beta-phenylethylamines.
Asunto(s)
Drogas Ilícitas/metabolismo , Oxazoles/metabolismo , Administración Oral , Animales , Estimulantes del Sistema Nervioso Central/metabolismo , Estimulantes del Sistema Nervioso Central/orina , Cromatografía Líquida de Alta Presión , Bromuro de Cianógeno/química , Estabilidad de Medicamentos , Hidrólisis , Hidroxilación , Drogas Ilícitas/orina , Masculino , Espectrometría de Masas , Oxazoles/orina , Oxidación-Reducción , Fenilpropanolamina/química , Ratas , Ratas Sprague-DawleyRESUMEN
The PM27 gene encodes a sea urchin skeletal protein. Both the transcript and encoded protein appear at the mesenchyme blastula stage and are restricted to the primary mesenchyme cell (PMC) lineage throughout development. Transgenic expression of PM27 promoter constructs demonstrates that this cell specificity is regulated at the level of transcription. The PM27 sequence predicts a nonglycosylated secretory product of 27 kDa in mature form. The N-terminal "repeat" domain of the deduced amino acid sequence consists of a series of tandem repeats and shares both sequence and predicted structural similarities with several fiber-forming proteins. The C-terminal "lectin" domain is similar to the C-class lectins. Antisera against either domain of PM27 detect two major proteins in embryo extracts, with apparent molecular weights of 27 and 30 kDa. Immunolocalization in whole embryos demonstrates that PM27 antigen is produced uniformly and exclusively by PMCs through the early prism stage and that this specificity is further restricted during skeletogenesis to a subpopulation of PMCs associated with the growing tips of the spicules. It is secreted to the skeletal compartment and accumulates predominantly at the advancing mineralizing surface of the spicule tips. As the spicules elongate PM27 protein disappears from the more mature mid-shaft regions. The observed characteristics of PM27 are consistent with a role in the regulation or execution of skeletal growth, as opposed to maintenance or structural integrity of the spicules.
Asunto(s)
Proteínas/química , Proteínas/genética , Erizos de Mar/crecimiento & desarrollo , Erizos de Mar/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sondas de ADN , Larva , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN/análisis , Alineación de SecuenciaRESUMEN
The crystals found in sporulation extracts of Bacillus thuringiensis (Berliner) contain proteins that are highly toxic to insects. Different crystal proteins exhibit distinct specificities for restricted groups of insects. An uncharacterized strain of B. thuringiensis (BtS2), derived from China, was found to carry several crystal protein genes and to be toxic to a wide variety of insects, including some coleopterans. Surprisingly, the coleopteran toxicity was traced to a CryIB-class protein. The previously cloned CryIB protein from B. thuringiensis ssp. thuringiensis strain HD-290-I, which was believed to be lepidopteran-specific, was also found to be toxic to at least two species of coleopteran larvae under certain conditions. In contrast to CryIB toxicity toward lepidopterans, the coleopteran activity of CryIB is enhanced by solubilization and by truncation with trypsin prior to administration. The magnitude of this effect varies with the host species and is reversed for the one lepidopteran tested. These results suggest that, for at least some insects, the apparent host specificity of CryIB may depend both on differences in midgut environment and on differences in toxin-receptor interaction. The results of insect toxicity experiments with a series of deletion mutants allowed definition of a CryIB protein fragment of ca. 65 kDa as the smallest peptide that retains bioactivity against both lepidopteran and coleopteran larvae. Deletions smaller than this resulted in the production of a protein that was nontoxic to both lepidopteran and coleopteran larvae.