RESUMEN

Phytases are widely used as feed additives for monogastric animals, which cannot easily utilise the phosphorus bound in phytate (myo-inositol hexakisphosphate). The current study presents a safety evaluation of a 6-phytase produced by an Aspergillus oryzae strain expressing two synthetic genes, both mimicking a phytase gene from a Citrobacter braakii strain. Oral administration of the phytase preparation to rats at a dose level of 0.86 g total organic solids/kg body weight/day for 13 weeks did not cause any adverse effect. The phytase preparation did not exhibit irritative potential when applied locally to the eyes of rabbits or when applied to the skin using the in vitro three-dimensional epidermis model of adult human-derived epidermal keratinocytes. Furthermore, the phytase preparation was found not to represent mutagenic or clastogenic potential in the bacterial reverse mutation assay and in the in vitro micronucleus assays. Based on the toxicological data, the large safety factors calculated under common recommended dose assumptions for broiler chickens and weaned piglets, and the fact that Aspergillus oryzae is considered a safe strain lineage, it is concluded that there are no reasons for safety concerns when using this phytase as a feed additive.

Asunto(s)

6-Fitasa/genética , 6-Fitasa/toxicidad , Aspergillus oryzae/genética , Aditivos Alimentarios/toxicidad , Genes Sintéticos , 6-Fitasa/biosíntesis , Alimentación Animal/efectos adversos , Animales , Aspergillus oryzae/enzimología , Pollos , Citrobacter/metabolismo , Ojo/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Conejos , Ratas , Piel/efectos de los fármacos , Porcinos

RESUMEN

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.

Asunto(s)

Proteínas de Unión al ADN/genética , Larva/genética , Proteínas Nucleares/genética , Urocordados/embriología , Urocordados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito , Datos de Secuencia Molecular , Factores de Transcripción/genética

RESUMEN

We have examined the expression and regulation of cytoskeletal actin genes in ascidians with tailed (Molgula oculata) and tailless larvae (Molgula occulta). Four cDNA clones were isolated representing two pairs of orthologous cytoskeletal actin genes (CA1 and CA2), which encode proteins differing by five amino acids in the tailed and tailless species. The CA1 and CA2 genes are present in one or two copies, although several related genes may also be present in both species. Maternal CA1 and CA2 mRNA is present in small oocytes but transcript levels later decline, suggesting a role in early oogenesis. In the tailed species, embryonic CA1 and CA2 mRNAs first appear in the presumptive mesenchyme and muscle cells during gastrulation, subsequently accumulate in the presumptive notochord cells, and can be detected in these tissues through the tadpole stage. CA1 mRNAs accumulate initially in the same tissues in the tailless species but subsequently disappear, in concert with the arrest of notochord and tail development. In contrast, CA2 mRNAs were not detected in embryos of the tailless species. Fertilization of eggs of the tailless species with sperm of the tailed species, which restores the notochord and the tail, also results in the upregulation of CA1 and CA2 gene expression in hybrid embryos. Antisense oligodeoxynucleotide experiments suggest that CA1 and CA2 expression in the notochord, but not in the muscle cells, is dependent on prior expression of Mocc FHI, an ascidian HNF-3beta-like gene. The expression of the CA1 and CA2 genes in the notochord in the tailed species, downregulation in the tailless species, upregulation in interspecific hybrids, and dependence on HNF-3beta activity is consistent with a role of these genes in development of the ascidian notochord.

Asunto(s)

Actinas/genética , Citoesqueleto/química , Proteínas de Unión al ADN/genética , Notocorda/crecimiento & desarrollo , Proteínas Nucleares/genética , Factores de Transcripción , Urocordados/crecimiento & desarrollo , Actinas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citoesqueleto/metabolismo , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Expresión Génica , Gónadas/embriología , Gónadas/metabolismo , Factor Nuclear 3-beta del Hepatocito , Datos de Secuencia Molecular , Urocordados/embriología

RESUMEN

Holoprosencephaly is a congenital defect of the median structures of the brain and face. The epidemiology is poorly known due to the paucity of population-based studies. This study describes the epidemiology of holoprosencephaly in a large population, using cases identified through the New York State Congenital Malformations Registry, and born in 1984-1989. We describe the craniofacial abnormalities present, their frequency, and their cooccurrence, and we examine the correspondence between the severity of craniofacial abnormalities, chromosomal abnormalities, and severity of the brain defect. Liveborn cases totaled 78, yielding a prevalence of 4.8 per 100,000 live births. Prevalence among girls was nearly double that in boys, and was 4.2 times higher among infants of mothers under age 18 compared to infants of older mothers. Only 9.8% of all cases had no craniofacial abnormalities other than the brain defect. Eye malformations were present in 76.8%, nose malformations in 69.5%, ear malformations in 50%, and oral clefts in 41.5%. These malformations arise at different times during gestation. The variability in patterns of cooccurrence suggests variability in the developmental pathways and/or timing of developmental derangements which result in holoprosencephaly. This, in turn, is consistent with a model of multiple causes. Children with alobar holoprosencephaly tended to have the most severe craniofacial anomalies, but the correspondence was not 100%. Craniofacial phenotype does not consistently discriminate between cytogenetically normal and abnormal cases.

Asunto(s)

Anomalías Craneofaciales/epidemiología , Holoprosencefalia/epidemiología , Aberraciones Cromosómicas/epidemiología , Trastornos de los Cromosomas , Anomalías del Ojo/epidemiología , Femenino , Variación Genética , Holoprosencefalia/mortalidad , Humanos , Lactante , Recién Nacido , Masculino , New York/epidemiología , Nariz/anomalías , Fenotipo , Prevalencia

RESUMEN

We have isolated a member of the HNF-3/forkhead gene family in ascidians as a means to determine the role of winged-helix genes in chordate development. The MocuFH1 gene, isolated from a Molgula oculata cDNA library, exhibits a forkhead DNA-binding domain most similar to zebrafish axial and rodent HNF-3beta. MocuFH1 is a single copy gene but there is at least one other related forkhead gene in the M. oculata genome. The MocuFH1 gene is expressed in the presumptive endoderm, mesenchyme and notochord cells beginning during the late cleavage stages. During gastrulation, MocuFH1 expression occurs in the prospective endoderm cells, which invaginate at the vegetal pole, and in the presumptive notochord and mesenchyme cells, which involute over the anterior and lateral lips of the blastopore, respectively. However, this gene is not expressed in the presumptive muscle cells, which involute over the posterior lip of the blastopore. MocuFH1 expression continues in the same cell lineages during neurulation and axis formation, however, during the tailbud stage, MocuFH1 is also expressed in ventral cells of the brain and spinal cord. The functional role of the MocuFH1 gene was studied using antisense oligodeoxynucleotides (ODNs), which transiently reduce MocuFH1 transcript levels during gastrulation. Embryos treated with antisense ODNs cleave normally and initiate gastrulation. However, gastrulation is incomplete, some of the endoderm and notochord cells do not enter the embryo and undergo subsequent movements, and axis formation is abnormal. In contrast, the prospective muscle cells, which do not express MocuFH1, undergo involution and later express muscle actin and acetylcholinesterase, markers of muscle cell differentiation. The results suggest that MocuFH1 is required for morphogenetic movements of the endoderm and notochord precursor cells during gastrulation and axis formation. The effects of inhibiting MocuFH1 expression on embryonic axis formation in ascidians are similar to those reported for knockout mutations of HNF-3beta in the mouse, suggesting that HNF-3/forkhead genes have an ancient and fundamental role in organizing the body plan in chordates.

Asunto(s)

Tipificación del Cuerpo , Proteínas de Unión al ADN/genética , Gástrula/fisiología , Proteínas Nucleares/genética , Factores de Transcripción/genética , Urocordados/embriología , Urocordados/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Linaje de la Célula , Movimiento Celular , Endodermo/citología , Endodermo/metabolismo , Factores de Transcripción Forkhead , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito , Larva/crecimiento & desarrollo , Larva/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Datos de Secuencia Molecular , Morfogénesis , Músculos/citología , Músculos/metabolismo , Notocorda/citología , Notocorda/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Oligonucleótidos Antisentido/farmacología , Factores de Transcripción/química , Factores de Transcripción/fisiología , Urocordados/crecimiento & desarrollo , Urocordados/metabolismo

RESUMEN

Our purpose was to examine trends over time in the use of prenatal cytogenetic diagnosis by New York State women from 1984 to 1993 in the context of new technologies in prenatal diagnosis and pregnancy management. Data are from the New York State Chromosome Registry and represent 95-98 per cent of all New York State women tested. Utilization rates were calculated separately for women under 35 years of age and for those aged 35 years and over. Analysis of longitudinal trends also utilized data from 1979 to 1983. Utilization by women under age 35 increased steadily over time but levelled off after 1986 among older women. Each year, there was an increase in the proportion of tests performed during the first trimester. The proportion of first-trimester tests performed with chorionic villus sampling (CVS) decreased from a maximum of 68 per cent in 1987 to 10 per cent in 1993. The percentage of tests performed due to 'advanced maternal age' decreased over time while 'suspected fetal pathology' categories increased in frequency. The increase in prenatal diagnosis tests performed because of abnormal maternal serum markers was associated with an increase over time in the number and proportion of Down syndrome fetuses detected prenatally and a slight decrease in the prevalence of Down syndrome among live births. The trends reported here parallel improvements in biochemical screening for Down syndrome and published suggestions that all women aged 30 and over are offered prenatal diagnosis.

Asunto(s)

Síndrome de Down/diagnóstico , Enfermedades Fetales/diagnóstico , Tamizaje Masivo/tendencias , Resultado del Embarazo , Diagnóstico Prenatal/tendencias , Adulto , Amniocentesis , Muestra de la Vellosidad Coriónica , Síndrome de Down/embriología , Síndrome de Down/prevención & control , Femenino , Enfermedades Fetales/embriología , Enfermedades Fetales/prevención & control , Humanos , Tamizaje Masivo/estadística & datos numéricos , New York , Embarazo , Diagnóstico Prenatal/métodos , Sistema de Registros

RESUMEN

The incidence of Down syndrome (DS) at conception is highly dependent on the maternal age distribution and age-specific pregnancy rates. The live birth prevalence of DS reflects these factors and fetal deaths. This study examined DS live birth prevalence from 1983 to 1992 in New York State and analysed the effects of demographic changes and prenatal diagnosis use on the observed live birth prevalence. Expected DS live birth prevalence without prenatal diagnosis was calculated and compared with observed. Data were obtained from birth defects registries, vital records, and population data maintained by the New York State Department of Health. Over time, DS live birth prevalence was stable at about 10.4 per 10,000 live births. The percentage and number of women in the population above age 30 increased, as did birth rates among these women. Birth rates among younger women decreased. The proportion of DS babies born to women aged 35 and over increased from 27.1 to 34.1 per cent. Use of prenatal diagnosis by this age group ranged from 39.6 to 43.2 per cent, and increased steadily from 1.8 to 4.3 per cent among women under 35. Detection of DS fetuses increased from 82 in 1985 to 233 in 1992. Without prenatal diagnosis, DS live birth prevalence in 1992 would have reached 15.3 per 10,000 live births compared to the 10.2 observed. Prenatal diagnosis has prevented an increase in DS live birth prevalence but has not been sufficient to reduce live birth prevalence significantly.

Asunto(s)

Síndrome de Down/epidemiología , Fertilidad , Edad Materna , Embarazo de Alto Riesgo , Diagnóstico Prenatal , Adolescente , Adulto , Negro o Afroamericano , Tasa de Natalidad , Síndrome de Down/diagnóstico , Femenino , Humanos , Persona de Mediana Edad , New York , Embarazo , Población Blanca

RESUMEN

This study examined the mortality experience of children with oral clefts using the New York State Congenital Malformations Registry. Infants born in the years 1983 to 1990 to New York residents, diagnosed with an oral cleft and matched to their birth certificate were included in the analysis. Children with oral clefts were compared to a sample of live births from the years 1983 to 1990 without malformations. Children with cleft palate without additional malformations had a statistically nonsignificant adjusted risk of 1.2 when compared to children with no malformations. Children with cleft lip with or without cleft palate had a 1.1 adjusted risk. However, 35% of children with oral clefts had associated malformations and experienced much higher mortality. Children with oral clefts should be carefully evaluated for additional malformations; if none are found, their mortality appears not to be elevated.

Asunto(s)

Fisura del Paladar/mortalidad , Anomalías Múltiples/mortalidad , Fisura del Paladar/complicaciones , Fisura del Paladar/etnología , Humanos , Lactante , Tablas de Vida , New York/epidemiología , Prevalencia , Sistema de Registros , Medición de Riesgo , Factores de Riesgo

RESUMEN

Cases in the New York State Congenital Malformations Registry are reported by hospitals and physicians. This study was undertaken to determine whether case finding should be expanded to include routine matching of Vital Records files to the registry in order to identify unreported children. Matching of children who were born in 1983-86 and who had a congenital malformation noted on their birth certificate yielded 2837 children who were not in the registry. The hospital of record was asked to submit a registry report if the child's medical record contained a congenital malformation. Medical records for 1267 (45%) of these children indicated that the child was normal, with no mention of a malformation. Medical records could not be located for 137. Registry reports were submitted for 1433, 67 of whom were subsequently found in the registry, leaving 1366 bona fide new cases. These new cases differ significantly from registry cases for a number of birth certificate variables and type of congenital malformation. The birth certificate cases were more likely than registry cases to have only one malformation and to have only a minor malformation. The 1366 new cases comprised 2.1% of all registry cases for 1983-86. Their addition increased the statewide prevalence of major malformations by 1.7% from 416.5 to 423.4 per 10 000 livebirths. Except for anencephaly, the prevalence of specific malformations was not altered measurably by the addition of these cases. Lengthy and continuous follow-up was required to obtain registry reports. The small number of cases found does not seem to justify the amount of resources that would be required to use birth certificates routinely to augment case finding in New York State.

Asunto(s)

Certificado de Nacimiento , Anomalías Congénitas/epidemiología , Registro Médico Coordinado , Sistema de Registros , Adolescente , Adulto , Distribución de Chi-Cuadrado , Niño , Anomalías Congénitas/clasificación , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Madres/estadística & datos numéricos , New York/epidemiología , Complicaciones del Trabajo de Parto/epidemiología , Oportunidad Relativa , Embarazo , Complicaciones del Embarazo/epidemiología , Sensibilidad y Especificidad

RESUMEN

Several but not all studies indicate that chorionic villus sampling (CVS) is associated with an increased risk for transverse limb deficiencies, including digital deficiencies. It has been suggested that variations in results regarding the transverse digital deficiencies (TDDs) may be due to the use of different classification criteria. We present the combined analysis of two case-control studies, the U.S. Multistate CVS (US) study and the Italian Multicentric Birth Defects (IP-IMC) study, using two different definitions of TDDs. We compared the frequency of CVS exposure in control infants with that among those infants with any number of affected digits (any TDD), and those with all five digits of at least one limb affected (extensive TDDs). The estimated relative risk (RR) for any TDD following CVS was 10.6 (IPIMC) and 6.6 (US). For the extensive TDDs, the RR was 30.5 (IPIMC) and 10.7 (US). In both studies, extensive TDDs were less than 25% of all TDDs. Compared to all TDDs, extensive TDDs were more likely to occur after CVS performed earlier in the first trimester (before 10-11 weeks' gestation). These findings suggest a relationship between the timing of CVS and the severity of TDDs; indicate that using a restrictive definition of TDDs (all five digits affected) may limit the ability to evaluate the association between CVS and TDDs in populations in whom CVS is usually performed at or after 10 weeks' gestation; and highlight the necessity to consider gestational age in any evaluation of the relative risk for limb deficiencies associated with CVS.

Asunto(s)

Muestra de la Vellosidad Coriónica , Adulto , Femenino , Edad Gestacional , Humanos , Embarazo

RESUMEN

OBJECTIVE: To evaluate the roles of 1) abdominal radiography, 2) a pressure diagnostic procedure (PDP) using a standardized diluent infusion into the catheter sideport, and 3) radiocontrast imaging of the catheter lumen as procedures for diagnosing catheter malfunction in diabetic patients implanted with a programmable intraperitoneal infusion device. RESEARCH DESIGN AND METHODS: Sixteen type I diabetic patients implanted with Infusaid programmable intraperitoneal insulin pumps were studied. The ability of the above three procedures to assist diagnosis of catheter malfunction and distinguish between occlusion and catheter breakage was retrospectively analyzed. Glycated hemoglobin was measured to determine the clinical importance of catheter malfunctions and decreases in pump flow due to insulin aggregation in the pump chamber. RESULTS: Mean glycated hemoglobin levels increased significantly from 8.0 +/- 0.3 to 9.0 +/- 0.4% (P < 0.05) before and after catheter malfunction, but not during pump flow slowdowns. Mean peak pressure during PDP was 1.96 +/- 0.14 psi (P < 0.01 vs. normal) in reversibly occluded catheters and 1.86 +/- 0.35 psi (P < 0.05 vs. normal) in broken catheters, compared with 1.32 +/- 0.23 psi in normal catheters. Decay times during PDP were > 50 s for both reversibly occluded and broken catheters (P < 0.001 vs. normal of 3.6 +/- 0.82 s). Abdominal radiographs and sideport injections of contrast material were used to distinguish the types of broken catheters. CONCLUSIONS: Catheter breakage and occlusion are complications in implantable insulin infusion systems and result in metabolic deterioration. The presence of a sideport allows pressure data and radiographic procedures to assist in determining the cause of catheter malfunction. A diagnostic algorithm was generated to improve efficiency in investigating catheter problems.

Asunto(s)

Catéteres de Permanencia/efectos adversos , Diabetes Mellitus Tipo 1/terapia , Diagnóstico por Imagen/métodos , Sistemas de Infusión de Insulina , Adulto , Glucemia , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/diagnóstico por imagen , Falla de Equipo , Femenino , Humanos , Insulina/sangre , Masculino , Persona de Mediana Edad , Radiografía , Estudios Retrospectivos

RESUMEN

Although numerous infants have been reported with transverse limb deficiencies after their mothers had undergone chorionic villus sampling (CVS), it has been unclear whether the procedure caused these defects. We report the results of the first multistate case-control study to assess and quantify the risk for specific limb deficiencies associated with CVS. Case subjects were 131 infants with nonsyndromic limb deficiency ascertained from 7 population-based birth defect surveillance programs, and born from 1988-1992 to mothers 34 years of age or older. Control subjects were 131 infants with other birth defects. We ascertained exposure to CVS from medical records and maternal and physician questionnaires. We assessed rates and timing of exposure to CVS, and estimated relative and absolute risks for anatomic subtypes of limb deficiency. The odds ratio for all types of limb deficiency after CVS from 8-12 weeks' gestation was 1.7 (95% confidence interval, 0.4-6.3). For specific anatomic subtypes, the strongest association was for transverse digital deficiency (odds ratio = 6.4; 95% confidence interval, 1.1-38.6). The risk for transverse digital deficiency increased with earlier gestational exposure (P < 0.01 for trend). We estimated that the absolute risk for transverse digital deficiency in infants after CVS was 1 per 2,900 births (0.03%). Exposure to CVS was associated with a sixfold increase in risk for transverse digital deficiency. The causality of this association is supported by its strength, specificity, biologic plausibility, and consistency with the results of previous studies. Although some centers already inform patients about risk for limb deficiency, this study quantifies the magnitude of risk associated with CVS from 8-12 weeks' gestation.

Asunto(s)

Muestra de la Vellosidad Coriónica/efectos adversos , Dedos/anomalías , Dedos del Pie/anomalías , Estudios de Casos y Controles , Anomalías Congénitas/epidemiología , Edad Gestacional , Humanos , Recién Nacido , Deformidades Congénitas de las Extremidades , Oportunidad Relativa , Método Simple Ciego , Estados Unidos/epidemiología

RESUMEN

Protein kinase C has been previously shown both to phosphorylate and to desensitize the ability of the human 5-HT1A receptor to inhibit adenylyl cyclase [Raymond, J. R. (1991) J. Biol. Chem. 266, 14747-14753]. In this study, we examined the effects of short-term treatment with protein kinase A activators on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the human serotonin 5-HT1A receptor in CHO cells that stably express 1200 fmol of receptor/mg of protein. Forskolin induced a concentration- and time-dependent phosphorylation of the receptor that was detectable at 5 min and maximal at 15-30 min with a half-maximal concentration of 10-20 microM. Phosphorylation was also induced by Sp-cAMPS or dibutyryl-cAMP, and blocked by Rp-cAMPS and a pseudosubstrate inhibitor of PKA, but not by heparin (inhibitor of receptor kinase) or sphingosine (inhibitor of PKC). The stoichiometry of phosphorylation induced by forskolin was 1 mol of phosphate per mole of receptor. PKA activators did not induce a measurable desensitization of 5-HT1A receptor-inhibited adenylyl cyclase activity. However, forskolin augmented the desensitization caused by a submaximal concentration of phorbol 12-myristate 13-acetate (300 nM PMA) as evidenced by a rightward shift of the concentration-response curve for 5-HT, and approximately doubled the amount of phosphate incorporated into the receptor by PMA. Forskolin did not augment desensitization or increase the degree of phosphorylation induced by a maximal concentration of PMA (5 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Serotonina/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Células CHO , Membrana Celular/metabolismo , Colforsina/farmacología , Cricetinae , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Heparina/farmacología , Humanos , Cinética , Fosforilación , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/metabolismo , Esfingosina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Tionucleótidos/farmacología , Transfección

RESUMEN

alpha 2A adrenergic receptors are thought to inhibit adenylyl cyclase primarily through Gi alpha 2. We tested the requirement for Gi alpha 2 to inhibit cAMP accumulation by stable expression of alpha 2A adrenergic receptors in mouse embryonic stem cells. Host lines consisted of wild-type CCE cells, and CCE cells with targeted disruption of the Gi alpha 2 gene by two-stage homologous recombination (Mortensen, R. M., Zubiuar, M., Neer, E. J., and Seidman, J. G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7036-7040; Mortensen, R. M., Conner, D. A., Chao, S., Geisterfer-Lowrance, A. A., and Seidman, J. G. (1992) Mol. Cell. Biol. 12, 2391-2395). Knockouts were confirmed by Northern blot and immunoblot. We studied three clones derived from wild-type CCE cells (2, 6, and 8) expressing 450 +/- 50, 3000 +/- 120, and 150 +/- 20 fmol of receptor/mg of protein, respectively, and two Gi alpha 2-null clones (7 and 18) expressing 2100 +/- 250 and 300 +/- 40 fmol of receptor/mg of protein. The specific agonist UK14304 caused an inhibition of cAMP accumulation in clones 2, 6 and 8 (58 +/- 16%, 62 +/- 7%, and 52 +/- 12%) and in clones 7 (47 +/- 3%) and 18 (40 +/- 5%), but not in nontransfected CCE cells. IC50 values were similar for all clones (approximately 200 nM). The effect was attenuated by pertussis toxin and the antagonist rauwolscine. These studies show that expression of Gi alpha 2 is not required for alpha 2A adrenergic receptors to inhibit cAMP accumulation.

Asunto(s)

AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos alfa 2/fisiología , Células Madre/metabolismo , Animales , Línea Celular , Embrión de Mamíferos/citología , Humanos , Ratones

RESUMEN

OBJECTIVE: To determine whether insulin antibodies are generated in diabetic patients after short- and long-term intraperitoneal insulin use and, if so, whether they are of potential clinical interest. Insulin antibodies commonly develop in diabetic patients who use subcutaneous human insulin, although their clinical significance remains controversial. Few data are available regarding insulin antibody responses to intraperitoneal insulin. RESEARCH DESIGN AND METHODS: We studied insulin antibody levels and clinical diabetes control in 25 type 1 diabetic patients treated for 3-6 years with intraperitoneal surfactant-stabilized porcine modified human insulin delivered by implantable programmable insulin delivery systems. RESULTS: All patients had preimplantation insulin antibody levels < 20 microU/ml, with a mean value of 2 +/- 2 microU/ml (1 SD). Mean antibody levels increased throughout the study period to a mean maximum of 197 +/- 326 microU/ml (P < 0.02) with 11 of 25 (44%) patients' levels exceeding 20 microU/ml (insulin responders). The mean time to significant antibody development was 21.8 +/- 4.4 months. Of the 11 responder patients, 4 had clinical syndromes that consisted of increasing daily insulin requirements and/or nocturnal hypoglycemia despite minimal nighttime basal insulin infusion rates associated with peak antibody levels > 200 microU/ml. None of the nonresponder patients (antibody levels < 20 microU/ml) had these clinical findings. CONCLUSIONS: Our results indicate that insulin antibody levels observed during intraperitoneal administration of human insulin are 1) similar to those reported during subcutaneous administration; although the rise in antibody level may be delayed compared with subcutaneous human insulin, 2) associated with a patient subset who are insulin antibody responders after switching from subcutaneous to intraperitoneal human insulin, 3) associated with a decrease in levels among responder patients regardless of whether they discontinue or continue pump use, and 4) associated with increased insulin needs and/or nocturnal hypoglycemia despite minimal basal rate insulin infusion at nighttime when antibody levels exceed 200 microU/ml.

Asunto(s)

Diabetes Mellitus Tipo 1/tratamiento farmacológico , Anticuerpos Insulínicos/biosíntesis , Sistemas de Infusión de Insulina , Insulina/uso terapéutico , Adulto , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inmunología , Femenino , Humanos , Inyecciones Intraperitoneales , Insulina/efectos adversos , Anticuerpos Insulínicos/sangre , Sistemas de Infusión de Insulina/efectos adversos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Tiempo

RESUMEN

OBJECTIVES: Since only short-term studies of continuous intraperitoneal insulin infusion (CIPII) therapy using implantable programmable insulin delivery systems have been performed to show this method of diabetes therapy to be safe and efficacious, we have performed long-term studies to assess its safety and efficacy. RESEARCH DESIGN AND METHODS: For 78 patient-years of follow-up, we have longitudinally studied the incidence of diabetic ketoacidosis and severe hypoglycemia in 25 type 1 diabetic patients treated with CIPII. We also compared, cross-sectionally, the long-term safety and efficacy of CIPII to intensive subcutaneous insulin therapy using intermittent injections or continuous subcutaneous insulin infusion. Finally, we examined the relationship between glycated hemoglobin levels and the standard deviation of daily blood glucose excursion. RESULTS: Cross-sectional analysis revealed similar degrees of metabolic control accompanied by significantly decreased rates of both ketoacidosis (0.013 events/patient/year) and severe hypoglycemia (0.05 events/patient/year) during CIPII compared to intermittent injections and continuous subcutaneous insulin infusion therapy. A four-fold decrease in the rate of severe hypoglycemia was observed during longitudinal comparison of pre- and post-implantation complication rates. A relationship was also shown between decreased levels of mean glycated hemoglobin and the standard deviation of blood glucose excursions during CIPII therapy. CONCLUSIONS: Our data demonstrate that long-term therapy with CIPII is as effective as other methods in achieving near-normal levels of glycated hemoglobin, which in CIPII is associated with a decreased standard deviation of blood glucose excursions. Further, CIPII using implantable programmable insulin delivery systems is the safest method described for intensive insulin therapy in home blood glucose monitoring type 1 diabetic patients.

Asunto(s)

Sistemas de Infusión de Insulina , Adolescente , Adulto , Anciano , Estudios Transversales , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Cetoacidosis Diabética , Femenino , Hemoglobina Glucada/análisis , Humanos , Hipoglucemia/etiología , Sistemas de Infusión de Insulina/efectos adversos , Estudios Longitudinales , Masculino , Persona de Mediana Edad

RESUMEN

We have studied the physical and functional linkages of heterologously expressed human 5-HT1A receptors to G protein alpha-subunits in HeLa and CHO-K1 cells. HeLa cells expressed immunoreactivity to G(i) proteins with an apparent rank order of G(i) alpha 3 (approximately 1 pmol/mg of protein) >> G(i) alpha 1 (approximately 0.1 pmol/mg) >> G(i) alpha 2 (< 0.02 pmol/mg), whereas CHO-K1 cells expressed immunoreactivity to G(i) alpha 2 (approximately 5 pmol/mg) >> G(i) alpha 3 (approximately 0.7 pmol/mg), but not to G(i) alpha 1. Both cell lines expressed large and small forms of Gs alpha, but neither expressed detectable G(o) alpha. Agonist-promotable physical coupling of the 5-HT1A receptor to G proteins was examined with high-affinity agonist binding and with co-immunoprecipitation using rabbit anti-receptor IgG fractions. Agonist treatment induced coupling of the 5-HT1A receptors to G proteins with an apparent rank order of G(i) alpha 3 > G(i) alpha 1, G(i) alpha 2 in HeLa cells and G(i) alpha 3 > G(i) alpha 2 in CHO-K1 cells. Agonist-promotable functional coupling of the 5-HT1A receptors to inhibition of adenylylcyclase was measured in membranes derived from HeLa and CHO-K1 cells expressing approximately 2.5-3 pmol of receptors/mg of protein by preincubation with antisera raised against the carboxyl termini of the G(i) protein alpha-subunits. A noteworthy difference between the two cell types was that antisera against the predominant G protein (G(i) alpha 2) were substantially more efficacious than G(i) alpha 3 antisera at blocking functional coupling to adenylylcyclase inhibition in CHO-K1 cells, whereas in HeLa cells, antisera against nonpredominant G proteins (G(i) alpha 1/G(i) alpha 2) were equally as effective as those against the predominant G protein (G(i) alpha 3). No physical or functional coupling of the 5-HT1A receptor to Gs alpha isoforms was detected in either cell line. These findings suggest that the 5-HT1A receptor can physically couple to multiple distinct G(i) proteins in mammalian cell membranes and that functional coupling to adenylylcyclase inhibition may be mediated by G(i) alpha 1, G(i) alpha 2, and G(i) alpha 3. One factor influencing the relative importance of those G proteins for 5-HT1A receptor-inhibited adenylylcyclase activity appears to be their-relative levels of expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Receptores de Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/metabolismo , Inhibidores de Adenilato Ciclasa , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Células CHO/metabolismo , Membrana Celular/metabolismo , Cricetinae , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/inmunología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Células HeLa/metabolismo , Humanos , Sueros Inmunes/inmunología , Immunoblotting , Técnicas de Inmunoadsorción , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Receptores de Serotonina/química , Receptores de Serotonina/inmunología

RESUMEN

D1 dopamine receptors stimulate cAMP accumulation in opossum kidney (OK) cells, but this response is attenuated by pretreatment with dopamine. Dopamine pretreatment also causes a reduction in D1 dopamine receptor number. We transfected OK cells with a rat cAMP phosphodiesterase cDNA (rPDE3) in order to determine the contribution of elevations of cAMP to those two phenomena. Wild-type (WT) OK cells were compared to three clones (C, H, and N) which demonstrated stable expression of the rPDE3 phenotype and genotype, rPDE3 RNA expression was confirmed in clones C, H, and N (but not in WT-OK cells) by reverse transcriptase-polymerase chain reaction. A functional rPDE3 phenotype was demonstrated in that dopamine-responsive cAMP accumulation was absent in clones C, H, and N in intact cells, but could be restored by preincubation with cAMP phosphodiesterase inhibitors, or by using washed membranes from those clones. All three clones had increased cAMP phosphodiesterase activity when compared to WT-OK cells (approximately 100% increase), and blunted or absent dopamine (1 microM)-induced protein kinase A activation. After pretreatment with dopamine (1 microM) for 1 h, clones C, H, and N desensitized equally well as WT-OK cells (approximately 40-50% reduction in maximal increase in cAMP). In contrast, down-regulation of D1 dopamine receptors was blunted for clone C (20% receptor loss) and absent for clones H and N, when compared to a 45% loss of receptors for WT-OK cells. These findings suggest that in OK cells pretreated with 1 microM dopamine (i) cAMP accumulation is not necessary for dopamine-induced desensitization, but (ii) is necessary for down-regulation of D1 dopamine receptors, and (iii) that the down-regulation and desensitization processes may be differentially regulated.

Asunto(s)

3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , AMP Cíclico/metabolismo , Riñón/metabolismo , Receptores de Dopamina D1/efectos de los fármacos , 3',5'-AMP Cíclico Fosfodiesterasas/genética , Adenilil Ciclasas/metabolismo , Animales , Células Cultivadas , Células Clonales , ADN , Depresión Química , Dopamina/farmacología , Regulación hacia Abajo , Activación Enzimática , Riñón/citología , Riñón/efectos de los fármacos , Zarigüeyas , Reacción en Cadena de la Polimerasa , Proteínas Quinasas/metabolismo , Ratas , Receptores de Dopamina D1/metabolismo

RESUMEN

Bovine pulmonary artery smooth muscle (SM) cells express a novel 5-hydroxytryptamine (5-HT) (5-HT4-like) receptor coupled to cAMP accumulation. cAMP radioimmunoassay established the agonist and antagonist profiles of this receptor. 5-HT (EC50 = 91 +/- 33 nM) and 5-methoxytryptamine were equipotent at the SM cell 5-HT receptor and both were more potent than 5-carboxamidotryptamine. Other tryptamine derivatives were less potent but remained full agonists. These findings are consistent with previous reports regarding 5-HT4 and 5-HT4-like receptors in the central nervous system. The most potent antagonists were the antidepressant compounds nortriptyline (IC50 = 177 +/- 153 nM) and zimelidine (IC50 = 202 +/- 101 nM). The 5-HT3 and 5-HT4 antagonist 3-tropanyl-indole-3-carboxylate (ICS 205-930) was also a competitive antagonist at this 5-HT4-like receptor (pA2 = 6.3). Antagonist affinities differed slightly at the SM cell receptor, compared with other 5-HT4 and 5-HT4-like receptors in the central nervous system. Nonetheless, the SM cell 5-HT4-like receptor displayed the same differential antagonist potencies as reported for these other receptors (ICS 205-930 > MDL 72222 and mianserin > ketanserin). 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was the most potent agonist for this 5-HT4-like receptor (EC50 = 6.4 +/- 3.4 nM). 8-OH-DPAT-induced cAMP accumulation could be blocked by ICS 205-930 but not by the 5-HT1A antagonist 1-(2-methoxyphenyl)-4-[4-(2-pthalimido)butyl]piperazine hydrobromide, distinguishing the SM cell 5-HT receptor from 5-HT1A receptors. The mechanism of 5-HT-stimulated cAMP production was also investigated. First, GTP augmented basal and 5-HT-stimulated cAMP accumulation. Second, antisera to the carboxyl terminus of the alpha subunit of Gs, attenuated 5-HT-mediated adenylate cyclase activation. This established that 5-HT-stimulated cAMP accumulation in SM cells required GS. These findings suggest that SM cells express a novel 5-HT4-like receptor positively coupled to adenylate cyclase. An unexpected finding was that 8-OH-DPAT is a potent partial agonist. These studies suggest that there may be heterogeneity among 5-HT4-like receptors.

Asunto(s)

Músculo Liso Vascular/metabolismo , Arteria Pulmonar/metabolismo , Receptores de Serotonina/biosíntesis , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Adenilil Ciclasas/metabolismo , Animales , Bovinos , Células Cultivadas , AMP Cíclico/metabolismo , Activación Enzimática , Hidrólisis , Indoles/farmacología , Fosfatidilinositoles/metabolismo , Radioinmunoensayo , Receptores de Serotonina/efectos de los fármacos , Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Tropisetrón , Fosfolipasas de Tipo C/metabolismo

RESUMEN

OBJECTIVE: This study was undertaken to learn about the involvement of optometrists in the eye care of diabetic patients in New York and to collect baseline data for planning intervention programs in diabetic eye disease. RESEARCH DESIGN AND METHODS: This study was conducted by a mail survey being sent to all optometrists in New York whom we could identify through state sources (n = 2270). Three follow-up mailings were conducted. RESULTS: Eighty-six percent who received our mailing responded, 87% of whom reported that they see diabetic patients. Analysis was confined to this 87%. Routine retinal examinations were provided by 68% of the respondents who see diabetic patients, 62% of whom dilate the pupils. Sixty-eight percent who see diabetic patients diagnose retinopathy solely by their own examination, and 60% of these dilate. Follow-up of retinopathy was provided by 15% of optometrists who see diabetic patients, 90% of whom dilate the pupils of their diabetic patients. Twenty-five percent of respondents who see diabetic patients refer them to an ophthalmologist for diagnosis of retinopathy, and 67% refer all retinopathy patients to an ophthalmologist for follow-up. Although 91% of respondents recommended eye examinations at least annually for diabetic patients, only 68% specifically recommended annual examinations through dilated pupils. Those who dilate were significantly more likely to recommend annual dilated examinations than those who do not dilate, regardless of practice setting. CONCLUSIONS: These findings indicate that optometrists as a group see many diabetic patients. Intervention strategies should include increased emphasis on the use of dilation when examining diabetic patients and annual dilated retinal examinations.

Asunto(s)

Retinopatía Diabética/diagnóstico , Optometría , Actitud del Personal de Salud , Ceguera/prevención & control , Estudios de Evaluación como Asunto , Estudios de Seguimiento , Humanos , Midriasis , New York , Derivación y Consulta , Encuestas y Cuestionarios