RESUMEN

Epigenetic mechanisms are increasingly understood to have major impacts across ecology. However, one molecular epigenetic mechanism, DNA methylation, currently dominates the literature. A second mechanism, histone modification, is likely important to ecologically relevant phenotypes and thus warrants investigation, especially because molecular interplay between methylation and histone acetylation can strongly affect gene expression. There are a limited number of histone acetylation studies on non-model organisms, yet those that exist show that it can impact gene expression and phenotypic plasticity. Wild birds provide an excellent system to investigate histone acetylation, as free-living individuals must rapidly adjust to environmental change. Here, we screen histone acetylation in the house sparrow (Passer domesticus); we studied this species because DNA methylation was important in the spread of this bird globally. This species has one of the broadest geographic distributions in the world, and part of this success is related to the way that it uses methylation to regulate its gene expression. Here, we verify that a commercially available assay that was developed for mammals can be used in house sparrows. We detected high variance in histone acetylation among individuals in both liver and spleen tissue. Further, house sparrows with higher epigenetic potential in the Toll Like Receptor-4 (TLR-4) promoter (i.e., CpG content) had higher histone acetylation in liver. Also, there was a negative correlation between histone acetylation in spleen and TLR-4 expression. In addition to validating a method for measuring histone acetylation in wild songbirds, this study also shows that histone acetylation is related to epigenetic potential and gene expression, adding a new study option for ecological epigenetics.

RESUMEN

Human activities are rapidly changing ecosystems around the world. These changes have widespread implications for the preservation of biodiversity, agricultural productivity, prevalence of zoonotic diseases, and sociopolitical conflict. To understand and improve the predictive capacity for these and other biological phenomena, some scientists are now relying on observatory networks, which are often composed of systems of sensors, teams of field researchers, and databases of abiotic and biotic measurements across multiple temporal and spatial scales. One well-known example is NEON, the US-based National Ecological Observatory Network. Although NEON and similar networks have informed studies of population, community, and ecosystem ecology for years, they have been minimally used by organismal biologists. NEON provides organismal biologists, in particular those interested in NEON's focal taxa, with an unprecedented opportunity to study phenomena such as range expansions, disease epidemics, invasive species colonization, macrophysiology, and other biological processes that fundamentally involve organismal variation. Here, we use NEON as an exemplar of the promise of observatory networks for understanding the causes and consequences of morphological, behavioral, molecular, and physiological variation among individual organisms.

RESUMEN

Glycoconjugate vaccines based on the Vi capsular polysaccharide directed against Salmonella enterica serovar Typhi are licensed or in development against typhoid fever, an important cause of morbidity and mortality in developing countries. Quantification of free polysaccharide in conjugate vaccines is an important quality control for release, to monitor vaccine stability and to ensure appropriate immune response. However, we found that existing separation methods based on size are not appropriate as free Vi non-specifically binds to unconjugated and conjugated protein. We developed a method based on free Vi separation by Capto Adhere resin and quantification by HPAEC-PAD. The method has been tested for conjugates of Vi derived from Citrobacter freundii with different carrier proteins such as CRM197, Tetanus Toxoid and Diphtheria Toxoid.

Asunto(s)

Cromatografía en Gel/métodos , Glicoconjugados/análisis , Polisacáridos Bacterianos/análisis , Salmonella typhi , Fiebre Tifoidea , Vacunas Tifoides-Paratifoides/análisis , Cromatografía Líquida de Alta Presión/métodos , Glicoconjugados/uso terapéutico , Humanos , Fiebre Tifoidea/prevención & control , Vacunas Tifoides-Paratifoides/uso terapéutico , Vacunas Conjugadas/análisis , Vacunas Conjugadas/uso terapéutico

RESUMEN

Salmonella paratyphi A is increasingly recognized as a common cause of enteric fever cases and there are no licensed vaccines against this infection. Antibodies directed against the O-polysaccharide of the lipopolysaccharide of Salmonella are protective and conjugation of the O-polysaccharide to a carrier protein represents a promising strategy for vaccine development. O-Acetylation of S. paratyphi A O-polysaccharide is considered important for the immunogenicity of S. paratyphi A conjugate vaccines. Here, as part of a programme to produce a bivalent conjugate vaccine against both S. typhi and S. paratyphi A diseases, we have fully elucidated the O-polysaccharide structure of S. paratyphi A by use of HPLC-SEC, HPAEC-PAD/CD, GLC, GLC-MS, 1D and 2D-NMR spectroscopy. In particular, chemical and NMR studies identified the presence of O-acetyl groups on C-2 and C-3 of rhamnose in the lipopolysaccharide repeating unit, at variance with previous reports of O-acetylation at a single position. Moreover HR-MAS NMR analysis performed directly on bacterial pellets from several strains of S. paratyphi A also showed O-acetylation on C-2 and C-3 of rhamnose, thus this pattern is common and not an artefact from O-polysaccharide purification. Conjugation of the O-polysaccharide to the carrier protein had little impact on O-acetylation and therefore should not adversely affect the immunogenicity of the vaccine.

Asunto(s)

Antígenos O/química , Polisacáridos Bacterianos/química , Ramnosa/química , Salmonella paratyphi A/inmunología , Acetilación , Conformación de Carbohidratos , Secuencia de Carbohidratos , Espectroscopía de Resonancia Magnética , Vacunas contra la Salmonella/química , Vacunas Conjugadas/química

RESUMEN

The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations.

Asunto(s)

Precipitación Química , Filtración , Antígenos O/aislamiento & purificación , Salmonella/metabolismo , Reactores Biológicos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Hidrólisis , Antígenos O/análisis , Antígenos O/metabolismo

RESUMEN

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.

Asunto(s)

Citrobacter/inmunología , Polisacáridos Bacterianos/aislamiento & purificación , Vacunas Tifoides-Paratifoides/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Citrobacter/química , Humanos , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/metabolismo , Tecnología Farmacéutica/métodos , Vacunas Tifoides-Paratifoides/química , Vacunas Tifoides-Paratifoides/metabolismo , Vacunas Conjugadas/química , Vacunas Conjugadas/metabolismo

RESUMEN

Introduced species offer unique opportunities to study evolution in new environments, and some provide opportunities for understanding the mechanisms underlying macroecological patterns. We sought to determine how introduction history impacted genetic diversity and differentiation of the house sparrow (Passer domesticus), one of the most broadly distributed bird species. We screened eight microsatellite loci in 316 individuals from 16 locations in the native and introduced ranges. Significant population structure occurred between native than introduced house sparrows. Introduced house sparrows were distinguished into one North American group and a highly differentiated Kenyan group. Genetic differentiation estimates identified a high magnitude of differentiation between Kenya and all other populations, but demonstrated that European and North American samples were differentiated too. Our results support previous claims that introduced North American populations likely had few source populations, and indicate house sparrows established populations after introduction. Genetic diversity also differed among native, introduced North American, and Kenyan populations with Kenyan birds being least diverse. In some cases, house sparrow populations appeared to maintain or recover genetic diversity relatively rapidly after range expansion (<50 years; Mexico and Panama), but in others (Kenya) the effect of introduction persisted over the same period. In both native and introduced populations, genetic diversity exhibited large-scale geographic patterns, increasing towards the equator. Such patterns of genetic diversity are concordant with two previously described models of genetic diversity, the latitudinal model and the species diversity model.

Asunto(s)

Variación Genética/genética , Gorriones/genética , Animales , Teorema de Bayes , Repeticiones de Microsatélite/genética , Gorriones/clasificación

RESUMEN

An efficacious, low cost vaccine against typhoid fever, especially for young children, would make a major impact on disease burden in developing countries. The virulence capsular polysaccharide of Salmonella Typhi (Vi) coupled to recombinant mutant Pseudomonas aeruginosa exoprotein A (Vi-rEPA) has been shown to be highly efficacious. We investigated the use of carrier proteins included in infant vaccines, standardized the conjugation process and developed key assays required for routine lot release at production scale. Vi from a BSL1 organism, Citrobacter freundii, strain WR7011, was used as an alternative to Vi from S. Typhi. We showed that Vi conjugated to CRM(197), a non-toxic mutant of diphtheria toxin, widely used in commercial vaccines, was produced at high yield. Vi-CRM(197) proved immunogenic in animal studies, even without adjuvant. Thus, Vi-CRM(197) appears to be a suitable candidate for the development of a commercially viable, effective typhoid vaccine for developing countries.

Asunto(s)

Adyuvantes Inmunológicos/administración & dosificación , Proteínas Bacterianas/administración & dosificación , Polisacáridos Bacterianos/inmunología , Vacunas contra Rickettsia/inmunología , Fiebre Tifoidea/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Citrobacter freundii/química , Citrobacter freundii/inmunología , Femenino , Inmunización Secundaria/métodos , Ratones , Ratones Endogámicos BALB C , Polisacáridos Bacterianos/administración & dosificación , Polisacáridos Bacterianos/aislamiento & purificación , Vacunas contra Rickettsia/administración & dosificación , Salmonella typhi/química , Salmonella typhi/inmunología , Fiebre Tifoidea/inmunología , Vacunación/métodos , Vacunas Conjugadas/administración & dosificación , Vacunas Conjugadas/inmunología

RESUMEN

Numerous studies have reported on enamel and dentine development in hominoid molars, although little is known about intraspecific incremental feature variation. Furthermore, a recent histological study suggested that there is little or no time between age at chimpanzee crown completion and age at molar eruption, which is unlikely given that root growth is necessary for tooth eruption. The study presented here redefines growth standards for chimpanzee molar teeth and examines variation in incremental features. The periodicity of Retzius lines in a relatively large sample was found to be 6 or 7 days. The number of Retzius lines and cuspal enamel thickness both vary within a cusp type, among cusps, and among molars, resulting in marked variation in formation time. Daily secretion rate is consistent within analogous cuspal zones (inner, middle, and outer enamel) within and among cusp types and among molar types. Significantly increasing trends are found from inner to outer cuspal enamel (3 to 5 microns/day). Cuspal initiation and completion sequences also vary, although sequences for mandibular molar cusps are more consistent. Cusp-specific formation time ranges from approximately 2 to 3 years, increasing from M1 to M2, and often decreasing from M2 to M3. These times are intermediate between radiographic studies and a previous histological study, although both formation time within cusps and overlap between molars vary considerably. Cusp-specific (coronal) extension rates range from approximately 4 to 9 microns/day, and root extension rates in the first 5 mm of roots range from 3 to 9 microns/day. These rates are greater in M1 than in M2 or M3, and they are greater in mandibular molars than in respective maxillary molars. This significant enlargement of comparative data on nonhuman primate incremental development demonstrates that developmental variation among cusp and molar types should be considered during interpretations and comparisons of small samples of fossil hominins and hominoids.

Asunto(s)

Evolución Biológica , Diente Molar/crecimiento & desarrollo , Pan troglodytes/crecimiento & desarrollo , Determinación de la Edad por los Dientes , Animales , Diente Canino/anatomía & histología , Diente Canino/crecimiento & desarrollo , Esmalte Dental/citología , Dentina/citología , Diente Molar/anatomía & histología , Paleodontología , Pan troglodytes/anatomía & histología , Pan troglodytes/genética , Corona del Diente/crecimiento & desarrollo , Erupción Dental

RESUMEN

Many vertebrates show seasonality in immune defenses, perhaps because of trade-offs with other physiological processes. Trade-offs between reproduction and immune function have been well studied, but how other life cycle events such as molt affect immune function remains unclear. Here, we hypothesize that one possible explanation is that accumulative dissociated processes (e.g., resource deficits generated over the long term by physiological processes) can have delayed effects on immune activity. To test this hypothesis, we compared cutaneous immune responses in groups of captive female house sparrows (Passer domesticus) photoperiodically induced into six different life cycle stages. We predicted that if delayed trade-offs occur, immune activity would be reduced after a mature life state was reached (e.g., postmolt) and not just compromised when other tissues were actively growing (instantaneous trade-off). We found evidence for both types of trade-offs: immune responses were weakest in sparrows that had just completed postnuptial molt, but they were also weak in birds growing reproductive tissues or feathers. Birds in mature reproductive states or light molt had strong immune responses comparable with birds in a nonbreeding/nonmolting state. Altogether, our results indicate that immune activity in female house sparrows can be influenced by both instantaneous and delayed trade-offs.

Asunto(s)

Muda/inmunología , Reproducción/inmunología , Piel/inmunología , Gorriones/inmunología , Animales , Femenino , Fotoperiodo , Estaciones del Año , Factores de Tiempo

RESUMEN

The analgesic effect of orally administered buprenorphine was compared with that induced by a standard therapeutic injected dose (0.05 mg/kg of body weight, s.c.) in male Long-Evans rats. Analgesia was assessed by measuring pain threshold, using the hot-water tail-flick assay before and after administration of buprenorphine. The results suggest that a commonly used formula for oral buprenorphine in flavored gelatin, at a dose of 0.5 mg/kg, does not increase pain threshold in rats. Instead, oral buprenorphine doses of 5 and 10 mg/kg were necessary to induce significant increases in pain threshold. However, these doses had to be administered by orogastric infusion because the rats would not voluntarily eat flavored gelatin containing this much buprenorphine. The depth of analgesia induced by these infused doses was comparable to that induced by the clinically effective s.c. treatment (0.05 mg/kg).

Asunto(s)

Analgésicos Opioides/administración & dosificación , Buprenorfina/administración & dosificación , Administración Oral , Analgesia , Animales , Gelatina , Masculino , Umbral del Dolor/efectos de los fármacos , Ratas , Ratas Long-Evans

RESUMEN

A vaccine trial was conducted with rhoptry-associated proteins 1 and 2 (RAP1 and RAP2) of Plasmodium falciparum in Saimiri boliviensis monkeys to compare the ability of parasite-derived (PfRAP1 and 2) and recombinant proteins (rRAP1 and 2) to induce protective immune responses and to find adjuvants suitable for use in humans. Eight groups of 6 monkeys each were immunized with parasite-derived or recombinant RAP1 and 2 with Freund's complete adjuvant (FCA) followed by Freund's incomplete adjuvant (FIA), Montanide ISA720 adjuvant, or CRL1005 adjuvant. Recombinant RAP1 and RAP2 were also administered separately, with Montanide ISA720. After 3 immunizations, monkeys were challenged by iv inoculation of 50,000 parasites of the Uganda Palo Alto strain of P. falciparum. Of the animals vaccinated using FCA/FIA, 1 of 6 control monkeys, 3 of 6 immunized with PfRAP1 and 2, and 2 of 6 with rRAP1 and 2 did not require drug treatment. Of the monkeys vaccinated with Montanide ISA720 adjuvant, 0 of the 6 control monkeys, 2 of 6 immunized with RAP1 and 2, 1 of 6 immunized with rRAP1, and 4 of 6 immunized with RAP2 did not require drug treatment. Two of 6 monkeys immunized with PfRAP1 and 2 with CRL1005 did not require treatment. All groups receiving RAP1, RAP2, or both had a significant decrease in initial parasite multiplication rates and there was a significant negative correlation between anti-RAP2 antibody and multiplication rates. Animals were rechallenged with the homologous parasite 126 days after the first challenge. Of the monkeys that did not require drug treatment after the first challenge, none developed detectable parasitemia following rechallenge.

Asunto(s)

Vacunas contra la Malaria , Malaria Falciparum/prevención & control , Parasitemia/prevención & control , Plasmodium falciparum/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Antiprotozoarios/biosíntesis , Anticuerpos Antiprotozoarios/sangre , Femenino , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/efectos adversos , Vacunas contra la Malaria/inmunología , Masculino , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Recombinantes/inmunología , Saimiri

RESUMEN

Several proteins expressed in Escherichia coli with the N-terminus Gly-Ser-Ser-[His]6- consisted partly (up to 20%) of material with 178 Da of excess mass, sometimes accompanied by a smaller fraction with an excess 258 Da. The preponderance of unmodified material excluded mutation, and the extra masses were attributed to posttranslational modifications. As both types of modified protein were N-terminally blocked, the alpha-amino group was modified in each case. Phosphatase treatment converted +258-Da protein into +178-Da protein. The modified His tags were isolated, and the mass of the +178-Da modification estimated as 178.06 +/- 0.02 Da by tandem mass spectrometry. As the main modification remained at +178 Da in 15N-substituted protein, it was deemed nitrogen-free and possibly carbohydrate-like. Limited periodate oxidations suggested that the +258-Da modification was acylation with a 6-phosphohexonic acid, and that the +178-Da modification resulted from its dephosphorylation. NMR spectra of cell-derived +178-Da His tag and synthetic alpha-N-d-gluconoyl-His tag were identical. Together, these results suggested that the +258-Da modification was addition of a 6-phosphogluconoyl group. A plausible mechanism was acylation by 6-phosphoglucono-1,5-lactone, produced from glucose 6-phosphate by glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Supporting this, treating a His-tagged protein with excess d-glucono-1,5-lactone gave only N-terminal gluconoylation.

Asunto(s)

Escherichia coli/metabolismo , Histidina/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Acilación , Secuencia de Aminoácidos , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Escherichia coli/genética , Gluconatos/metabolismo , Humanos , Técnicas In Vitro , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteína Tirosina Quinasa ZAP-70 , Quinasas de Receptores Adrenérgicos beta

RESUMEN

OBJECTIVE: Our purpose was to determine whether the femur length-to-abdominal circumference ratio can be used antenatally to predict a lethal skeletal dysplasia. STUDY DESIGN: All obstetric sonograms performed from January 1990 to October 1995 were reviewed (44,020 studies) to find those scans suggestive of a skeletal dysplasia. Thirty patients were identified. The femur length/abdominal circumference ratio was then calculated from each patient's initial and subsequent sonograms. Birth outcomes were obtained on the 27 patients who elected to continue their pregnancies. RESULTS: All fetuses with a lethal skeletal dysplasia (n = 12) had a ratio <0.16. The fetuses with a nonlethal dysplasia (n = 8) had ratios between 0.134 and 0.193, with only 1 fetus with a ratio <0.16. All fetuses with no evidence of a skeletal dysplasia after birth (n = 7) had femur length/abdominal circumference ratios >0.18. The 1 fetus with a ratio <0.16 who survived the neonatal period had extreme bowing and demonstrates the limitation of the ratio when bowing is present. CONCLUSIONS: A stillbirth or neonatal death occurred in 12 of 13 patients with a femur length/abdominal circumference ratio <0.16, independent of gestational age. Conversely, no fetus with a ratio >0.16 was found to have a lethal skeletal dysplasia. This information may be useful in counseling women when ultrasonography suggests the diagnosis of a skeletal dysplasia.

Asunto(s)

Abdomen/diagnóstico por imagen , Enfermedades del Desarrollo Óseo/diagnóstico por imagen , Fémur/diagnóstico por imagen , Resultado del Embarazo , Ultrasonografía Prenatal , Enfermedades del Desarrollo Óseo/mortalidad , Femenino , Muerte Fetal/epidemiología , Predicción , Humanos , Incidencia , Mortalidad Infantil , Recién Nacido , Embarazo , Radiografía

RESUMEN

The carboxy terminus of GLUT-4 contains a functional internalization motif (Leu-489Leu-490) that helps maintain its intracellular distribution in basal adipocytes. This motif is flanked by the major phosphorylation site in this protein (Ser-488), which may play a role in regulating GLUT-4 trafficking in adipocytes. In the present study, the targeting of GLUT-4 in which Ser-488 has been mutated to alanine (SAG) has been examined in stably transfected 3T3-L1 adipocytes. The trafficking of SAG was not significantly different from that of GLUT-4 in several respects. First, in the absence of insulin, the distribution of SAG was similar to GLUT-4 in that it was largely excluded from the cell surface and was enriched in small intracellular vesicles. Second, SAG exhibited insulin-dependent movement to the plasma membrane (4- to 5-fold) comparable to GLUT-4 (4- to 5-fold). Finally, okadaic acid, which has previously been shown to stimulate both GLUT-4 translocation and its phosphorylation at Ser-488, also stimulated the movement of SAG to the cell surface similarly to GLUT-4. Using immunoelectron microscopy, we have shown that GLUT-4 is localized to intracellular vesicles containing the Golgi-derived gamma-adaptin subunit of AP-1 and that this localization is enhanced when Ser-488 is mutated to alanine. We conclude that the carboxy-terminal phosphorylation site in GLUT-4 (Ser-488) may play a role in intracellular sorting at the trans-Golgi network but does not play a major role in the regulated movement of GLUT-4 to the plasma membrane in 3T3-L1 adipocytes.

Asunto(s)

Adipocitos/metabolismo , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas del Tejido Nervioso , Células 3T3 , Alanina , Secuencia de Aminoácidos , Animales , Transportador de Glucosa de Tipo 3 , Transportador de Glucosa de Tipo 4 , Humanos , Leucina , Ratones , Proteínas de Transporte de Monosacáridos/biosíntesis , Mutagénesis Sitio-Dirigida , Fosforilación , Mutación Puntual , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Serina , Transfección

RESUMEN

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.

Asunto(s)

Adipocitos/metabolismo , Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Células 3T3 , Animales , Transporte Biológico Activo , Western Blotting , Transportador de Glucosa de Tipo 4 , Glutatión Transferasa/metabolismo , Sustancias Macromoleculares , Ratones , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Dodecil Sulfato de Sodio , Propiedades de Superficie , Tensoactivos

RESUMEN

Vesicle-associated membrane protein 2 (VAMP2) has been implicated in the insulin-regulated trafficking of GLUT4 in adipocytes. It has been proposed that VAMP2 co-localizes with GLUT4 in a postendocytic storage compartment (Martin, S., Tellam, J., Livingstone, C., Slot, J. W., Gould, G. W., and James, D. E. (1996) J. Cell Biol. 134, 625-635), suggesting that it may play a role distinct from endosomal v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) such as cellubrevin that are also expressed in adipocytes. The present study examines the effects of recombinant glutathione S-transferase (GST) fusion proteins encompassing the entire cytoplasmic tails of VAMP1, VAMP2, and cellubrevin on insulin-stimulated GLUT4 translocation in streptolysin O permeabilized 3T3-L1 adipocytes. GST-VAMP2 inhibited insulin-stimulated GLUT4 translocation by approximately 35%, whereas GST-VAMP1 and GST-cellubrevin were without effect. A synthetic peptide corresponding to the unique N terminus of VAMP2 also inhibited insulin-stimulated GLUT4 translocation in a dose-dependent manner. This peptide had no effect on either guanosine 5'-3-O-(thio)triphosphate-stimulated GLUT4 translocation or on insulin-stimulated GLUT1 translocation. These results imply that GLUT4 and GLUT1 may undergo insulin-stimulated translocation to the cell surface from separate intracellular compartments. To confirm this, adipocytes were incubated with a transferrin-horseradish peroxidase conjugate to fill the itinerant endocytic system after which cells were incubated with H2O2 and diaminobenzidine. This treatment completely blocked insulin-stimulated movement of GLUT1, whereas in the case of GLUT4, movement to the surface was delayed but still reached similar levels to that observed in insulin-stimulated control cells after 30 min. These results suggest that the N terminus of VAMP2 plays a unique role in the insulin-dependent recruitment of GLUT4 from its intracellular storage compartment to the cell surface.

Asunto(s)

Adipocitos/metabolismo , Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas del Tejido Nervioso/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Aplysia , Compartimento Celular , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Ratones , Datos de Secuencia Molecular , Proteínas R-SNARE , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Proteína 3 de Membrana Asociada a Vesículas

RESUMEN

We sought to determine the sensitivity of detecting congenital heart defects with routine antenatal ultrasound including the four-chamber view in a population at low risk for cardiac anomalies. Neonatal outcome and anomaly databases were reviewed to identify cases of morphologic cardiac defects from 1988-1992. Of 176 cases identified, 62 (35%) had routine antenatal ultrasound including the four-chamber view. Thirteen of these 62 (21%) were diagnosed by ultrasound and 18 of the 116 (15%) total defects present were seen. Of these, only 16 of 25 (64%) defects reasonably expected to be seen by the four-chamber cardiac view alone were detected. The routine ultrasound four-chamber cardiac evaluation is limited and detection depends on the type of cardiac defect present.

Asunto(s)

Ecocardiografía , Atrios Cardíacos/diagnóstico por imagen , Cardiopatías Congénitas/diagnóstico por imagen , Ventrículos Cardíacos/diagnóstico por imagen , Ultrasonografía Prenatal , Femenino , Estudios de Seguimiento , Edad Gestacional , Atrios Cardíacos/embriología , Cardiopatías Congénitas/embriología , Cardiopatías Congénitas/epidemiología , Ventrículos Cardíacos/embriología , Humanos , Incidencia , Recién Nacido , Embarazo , Estudios Retrospectivos , Sensibilidad y Especificidad

RESUMEN

OBJECTIVE: To evaluate standardized developmental test performance of infants and children who as fetuses had mild isolated cerebral ventriculomegaly diagnosed by ultrasound. METHODS: Ultrasound records from 1990 to 1996 were searched for cases of mild isolated ventriculomegaly, and standardized developmental testing of the children was offered to their parents. Each consented child was matched to a normal antepartum subject with respect to sex, race, indication for ultrasound, and gestational age (+/- 2 weeks) at the time of ultrasound. Tests of cognitive, motor, and adaptive behavior were then administered by examiners blinded to the subjects' case or comparison status. RESULTS: Twenty-two cases and an equal number of matched comparison subjects completed the testing. The ventriculomegaly and comparison groups were similar with respect to parental age, maternal education, and household income. The ventriculomegaly subjects scored significantly lower than the comparison group on both the Bayley Scales of Infant Development: mental development index (88.95 versus 99.68, P = .017) and psychomotor development index (95.99 versus 103.95, P = .039). Eight of the 22 ventriculomegaly children were classified as developmentally delayed on the mental developmental index compared with one of 22 children in the comparison group (P = .021). Adaptive behavior skills, as measured by the Vineland Behavior Scales (99.64 versus 102.68), were not significantly different between the groups (P = .571). CONCLUSION: Mild isolated ventriculomegaly detected on antepartum sonographic examination is associated with a significant risk for developmental delay. Insofar as these children were judged to be completely normal at birth, our findings represent an important application of antepartum sonography for identifying infants who could be targeted for early childhood intervention.

Asunto(s)

Ventrículos Cerebrales/patología , Desarrollo Infantil , Ventrículos Cerebrales/diagnóstico por imagen , Preescolar , Ecoencefalografía , Femenino , Estudios de Seguimiento , Humanos , Lactante , Embarazo , Ultrasonografía Prenatal

RESUMEN

For over 100 years, the eosinophil has been associated with allergic disease. At present, eosinophils appear to be associated pathologically with asthma, atopic dermatitis, allergic rhinitis, eosinophilic gastroenteritis, and certain eye diseases. The effector functions of eosinophils appear to be derived primarily from release of lipid mediators and proteins, including cytokines and granule proteins. Eosinophil degranulation results in the release of several cytotoxic cationic granule proteins. Furthermore, release of cytokines by eosinophils and other cells involved in inflammation amplifies and regulates localized immune responses. Altogether, the eosinophil's capacity to release and be influenced by a variety of mediators, including the granule proteins and cytokines, implicates this cell in the pathology of inflammation and in the perpetuation of the inflammatory response.

Asunto(s)

Degranulación de la Célula/inmunología , Citocinas/sangre , Eosinófilos/inmunología , Hipersensibilidad/sangre , Eosinófilos/metabolismo , Eosinófilos/patología , Humanos