RESUMO
The effects of ovariectomy on the growth of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors were investigated after rats ahd been exposed prenatally to diethylstilbestrol (DES). Pregnant rats were inoculated with either DES (total dose: 1.2 micrograms) in sesame oil or with the vehicle alone on days 10 and 13 of gestation. Female offspring were given 2 gastric intubations of DMBA (10 mg each) 1 week apart beginning at 50 plus or minus 1 days of age. When the average diameter of a mammary tumor exceeded 2 cm, the animal was ovariectomized. The initial response of most tumors in both the DES-exposed and control groups to ovariectomy was size regression. The growth of 7 tumors that arose soon after DMBA treatment in each group was studied for 12-20 weeks after ovariectomy. Whereas only 1 tumor from the control group resumed active growth after the initial regression period, 6 tumors in the DES-exposed group overcame the initial effects of ovariectomy and began to grow again. Thus ovariectomy appeared to be less effective in producing sustained control growth in DMBA-induced mammary tumors in rats exposed prenatally to DES.
Assuntos
Dietilestilbestrol , Neoplasias Mamárias Experimentais/induzido quimicamente , Troca Materno-Fetal , Ovário/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Adenocarcinoma/induzido quimicamente , Adenofibroma/induzido quimicamente , Animais , Castração , Feminino , Neoplasias Mamárias Experimentais/patologia , Gravidez , Prognóstico , Ratos , Fatores de TempoRESUMO
Aspects of the development, morphology, and estrogen binding capacity of mammary tumors in rats exposed prenatally to the synthetic estrogen, diethylstilbestrol (DES), and treated postnatally with 7,12-dimethylbenz(a)anthracene (DMBA) were analyzed as part of a project aimed at understanding the effects of transplacental exposure to DES on estrogen-sensitive tissues. Pregnant Sprague-Dawley rats were given injections of DES (total dose, 1.2 micrograms) or vehicle alone on Days 15 and 18 of gestation. All female offspring were given gastric intubations of DMBA, either a single 10-mg dose on Day 50 or two doses (10 mg each) on Days 50 and 57. Among rats treated postnatally with 10 mg of DMBA, the DES-exposed group had a significantly greater incidence of palpable mammary tumors than did the vehicle-exposed controls. In addition, there was an earlier time of appearance of palpable tumors in the DES-exposed group. When the data from rats treated postnatally with two 10-mg doses of DMBA were analyzed, there were no significant differences in palpable mammary tumor incidence or tumor latency between the DES-exposed and vehicle-exposed groups. When the pathology of the mammary tumors produced in rats treated with 10 mg of DMBA was analyzed, the DES-exposed group had a significantly higher proportion of benign tumors (fibroadenoma, adenoma, lobular hyperplasia) than adenocarcinomata compared to vehicle-exposed controls. Both exposure groups had similar numbers of nonpalpable mammary lesions discovered at necropsy. Estrogen binding capacities of representative adenocarcinomata did not differ significantly between the two prenatal exposure groups treated postnatally with 10 mg of DMBA. These results demonstrate the importance of the dose of the challenge carcinogen in revealing the effects of transplacental drug exposure and may have special significance for women who were exposed to DES in utero.
Assuntos
9,10-Dimetil-1,2-benzantraceno , Benzo(a)Antracenos , Dietilestilbestrol/farmacologia , Neoplasias Mamárias Experimentais/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal , Animais , Estradiol/metabolismo , Feminino , Neoplasias Mamárias Experimentais/patologia , Gravidez , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
Female ACI rats were exposed to diethylstilbestrol (DES) transplacentally and followed to 10 months of age to assess the effect of the drug on mammary development and tumorigenesis. Pregnant rats were given injections of vehicle (sesame oil) or DES (total dose, 0.8 micrograms = low DES or 8.0 micrograms = high DES) on days 15 and 18 of gestation. Pellets containing 2.5 mg DES + 17.5 mg cholesterol (DES pellet) or 20 mg cholesterol (chol pellet) were implanted s.c. into 12-week-old female offspring, creating 6 experimental groups: vehicle exposure + chol pellet (1) or + DES pellet (2); low DES exposure + chol pellet (3) or + DES pellet (4); high DES exposure + chol pellet (5) or + DES pellet (6). At sacrifice, representative mammary tissue and all palpable mammary tumors were removed for histopathological analysis. Each of the 6 experimental groups contained a minimum of 32 rats from at least 14 litters. In computation of data, the unit of analysis was the litter. Groups which had received any DES (prenatally or postnatally) were found to have elongated nipples and enlarged pituitaries. The mammary gland whole mounts from all rats in groups 4 and 6 displayed extensive lobuloalveolar proliferation comparable to that seen in DES pellet controls (group 2). Mammary glands of approximately 75% of rats in groups 3 and 5 were categorized as showing the lowest grade of differentiation while this undifferentiated condition was seen in only 36% of group I controls. No palpable mammary tumors were found in rats exposed to vehicle in utero (group 1). But in group 5, a total of 6 tumors in 5 animals derived from 4 different litters were obtained, a difference shown to be statistically significant. Group 3 had 1 rat with 8 tumors. Among rats bearing the DES pellet, tumor latency was shortened significantly in both groups exposed to DES in utero. By 22 weeks after pellet implantation, 100% of the DES-exposed litters (groups 4 and 6) contained at least 1 tumor-bearing rat compared to about 50% of the tumor-bearing litters in group 2. Tumor multiplicity at sacrifice was increased significantly in the group exposed prenatally to the higher dose of DES. Histologically, the overwhelming majority of palpable mammary tumors from all tumor-bearing treatment groups were classified as adenocarcinomas. Prenatal exposure to DES did not alter the ratio of malignant to benign lesions observed, nor did it affect the degree of differentiation noted in the adenocarcinomas.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Dietilestilbestrol/toxicidade , Glândulas Mamárias Animais/efeitos dos fármacos , Neoplasias Mamárias Experimentais/induzido quimicamente , Troca Materno-Fetal , Efeitos Tardios da Exposição Pré-Natal , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/patologia , Animais , Feminino , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Mutação , Tamanho do Órgão , Hipófise/patologia , Gravidez , Prolactina/sangue , Ratos , Ratos Endogâmicos ACI , Fatores de TempoRESUMO
Regulation of cyclic AMP through its synthesis is known to be important in modulating the activity of molluscan neurons; however, no data exists regarding the regulation of cyclic AMP degradation. We find that cyclic AMP phosphodiesterase (PDE) activity in homogenates of the nervous system of the mollusc Pleurobranchaea is significantly stimulated by calcium ion. Ca2+ stimulation is suppressed by the calmodulin antagonist trifluoperazine (TFP), indicating resemblance to the Ca2+-calmodulin PDEs of mammalian neurons. Ca2+ also accentuates the pH sensitivity of PDE. The qualities of Ca2+ and pH sensitivity of PDE are fitted into a model for cAMP regulation of neuronal activity in an identified feeding command neuron; the postulated role of PDE is consistent with effects of cAMP, TFP, and pH on the neuron's activity.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Cálcio/metabolismo , Sistema Nervoso/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Calmodulina/metabolismo , Ativação Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Moluscos , Trifluoperazina/farmacologiaRESUMO
Genital tract morphology in 14-month old female rats exposed prenatally to diethylstilbestrol (DES) was analyzed as part of an examination of the effects of transplacental exposure to DES on estrogen sensitive tissues. Pregnant Sprague-Dawley rats were injected with sesame oil alone or with DES in sesame oil on days 10 and 13 of gestation (total dose 1.2 micrograms DES) or on days 15 and 18 (total dose 1.2 micrograms or 120 micrograms DES). Female offspring (9-15 per group) were sacrificed at 14 months of age. Effects of DES exposure varied with the dose given and with the stage of differentiation of the fetal tissues. In the ovaries of rats exposed to 120 micrograms of DES on days 15 and 18 of gestation, follicular elements were reduced and replaced by dense sheets of stromal cells; oophoritis was noted in five of nine rats. Hypercellularity of oviductal stroma was another common feature, as was suppurative salpingitis. Ovaries of rats exposed to 1.2 micrograms DES on days 10 and 13 of gestation were more likely to contain numerous corpora lutea than the other DES-exposed groups of controls. An increased incidence of benign uterine abnormalities was observed in DES-exposed offspring, including squamous metaplasia and suppurative endometritis. In the cervices of all nine rats exposed to 120 micrograms DES on days 15 and 18 of gestation, the epithelial surface showed a convoluted pattern, lined by stratified squamous and stratified cuboidal cells. Thus, prenatal exposure to DES, especially at the higher dose used, has long-term consequences on reproductive tract morphology in Sprague-Dawley rats.
Assuntos
Anormalidades Induzidas por Medicamentos/patologia , Dietilestilbestrol/efeitos adversos , Genitália Feminina/anormalidades , Efeitos Tardios da Exposição Pré-Natal , Animais , Doenças das Tubas Uterinas/induzido quimicamente , Doenças das Tubas Uterinas/patologia , Feminino , Doenças Ovarianas/induzido quimicamente , Doenças Ovarianas/patologia , Gravidez , Resultado da Gravidez , Ratos , Ratos Endogâmicos , Doenças do Colo do Útero/induzido quimicamente , Doenças do Colo do Útero/patologia , Doenças Uterinas/induzido quimicamente , Doenças Uterinas/patologia , Doenças Vaginais/induzido quimicamente , Doenças Vaginais/patologiaRESUMO
BACKGROUND/PURPOSE: The T-tube ileostomy was first used at Texas Children's Hospital in 1959. The purpose of this study is to update the experience since the initial report of this technique in 1981. METHODS: A database of 448 patients with cystic fibrosis (CF) seen in the authors' institution was used to identify 83 patients (18.5%) who presented with meconium ileus. The clinic and hospital charts of these patients were reviewed retrospectively to identify patients who had undergone placement of a T-tube ileostomy. RESULTS: Surgery was performed in 60 of 83 patients for complications of meconium ileus or failure to evacuate the meconium after a contrast enema. Of these patients, 21 of 60 (35%) underwent placement of a T-tube ileostomy. An additional 8 patients were identified who underwent placement of a T-tube ileostomy but were not included in the CF database, for a total of 29 patients who have been treated with T-tube ileostomy since 1959 at Texas Children's Hospital. Five patients were excluded from analysis because of insufficient data or misdiagnosis. One of the 24 patients in the series died of complications of prematurity. A total of 20 of 23 patients had resolution of their meconium ileus after T-tube irrigation with n-acetylcysteine or pancreatic enzymes. Three patients required additional surgery to relieve persistent bowel obstruction. All patients had the T-tube removed within the first 8 weeks after surgery. Two patients required subsequent repair of an incisional hernia. There were otherwise no complications of this procedure, with an average follow-up of 11.5 years. CONCLUSION: In patients with uncomplicated meconium ileus unrelieved by contrast enema, the T-tube ileostomy is an effective and safe treatment.
Assuntos
Fibrose Cística/complicações , Ileostomia/métodos , Obstrução Intestinal/cirurgia , Humanos , Recém-Nascido , Obstrução Intestinal/etiologia , MecônioRESUMO
Six foods representing a variety of food products were analyzed by the Assurance Listeria polyclonal enzyme immunoassay (EIA) and by either the Bacteriological Analytical Manual or the U.S. Department of Agriculture culture method for detecting Listeria monocytogenes and related Listeria species. Samples of each food type, at each inoculation level, were analyzed simultaneously by both methods. A total of 19 laboratories representing federal government agencies and private industry in the United States and Canada participated. Food types were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots of green beans, which were naturally contaminated. During this study, 1764 samples and controls were analyzed and confirmed, of which 492 were positive and 947 were negative by both methods. There were 159 samples that were positive by culture method but negative by the EIA and 188 that were negative by culture method but positive by EIA. Twenty-two samples were negative by EIA and by culture method but confirmed positive when Assurance selective enrichment broths were subcultured to selective agar. The Assurance polyclonal EIA for detecting L. monocytogenes and related Listeria species in foods has been adopted first action by AOAC INTERNATIONAL.
Assuntos
Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Listeria/isolamento & purificação , Animais , Anticorpos , Canadá , Bovinos , Distribuição de Qui-Quadrado , Meios de Cultura , Laticínios/microbiologia , Decápodes , Fabaceae/microbiologia , Guias como Assunto , Técnicas Imunoenzimáticas , Listeria/imunologia , Listeria/metabolismo , Listeria monocytogenes/imunologia , Listeria monocytogenes/metabolismo , Produtos da Carne/microbiologia , Plantas Medicinais , Produtos Avícolas/microbiologia , Controle de Qualidade , Padrões de Referência , Tamanho da Amostra , Frutos do Mar/microbiologia , Espectrofotometria Ultravioleta , Suínos , Estados UnidosRESUMO
Six foods representing a variety of food products were analyzed by the Visual Immunoprecipitate Assay (VIP) and either the Bacteriological Analytical Manual (BAM) or the U.S. Department of Agriculture culture methods for detection of Listeria monocytogenes and related Listeria spp. Samples of each food type at each inoculation level were simultaneously analyzed by both methods. A total of 23 laboratories representing federal agencies and private industry in the United States and Canada participated in this collaborative study. Foods were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots of green beans that were naturally contaminated. During this study, 1509 samples and controls were analyzed and confirmed, of which 370 were positive and 921 were negative by both methods. One hundred and fifteen samples were positive by culture methods but negative by VIP. One hundred and thirty-two were negative by culture methods but positive by the VIP. Twenty-nine samples were negative by VIP and by culture methods but confirmed positive when VIP selective enrichment broths were subcultured to selective agars. The VIP method for detection of L. monocytogenes and related Listeria spp. in foods has been adopted first action by AOAC INTERNATIONAL.
Assuntos
Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Listeria/isolamento & purificação , Animais , Canadá , Bovinos , Distribuição de Qui-Quadrado , Meios de Cultura , Laticínios/microbiologia , Decápodes , Fabaceae/microbiologia , Guias como Assunto , Listeria/imunologia , Listeria monocytogenes/imunologia , Produtos da Carne/microbiologia , Plantas Medicinais , Produtos Avícolas/microbiologia , Testes de Precipitina , Tamanho da Amostra , Frutos do Mar/microbiologia , Estados Unidos , United States Department of AgricultureAssuntos
Dietilestilbestrol/farmacologia , Genitália Feminina/efeitos dos fármacos , Hormônios/sangue , Glândulas Mamárias Animais/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , 9,10-Dimetil-1,2-benzantraceno/farmacologia , Animais , Estrogênios/sangue , Feminino , Tamanho do Órgão/efeitos dos fármacos , Gravidez , Progesterona/sangue , Prolactina/sangue , RatosRESUMO
Selection for survivors percent egg production from first egg to 40 weeks of age was conducted for six generations. Within-line selection (WLS) on the basis of an index of individual records plus sire family and dam family means was compared with reciprocal recurrent selection (RRS) based on sire family selection among cross progeny. Genetically heterogeneous synthetic populations, the Cornell Control and the Purdue Pool strains were used.The responses to WLS (3.84 °) and RRS (2.57 °) were both significantly greater than zero, but were not significantly different from each other. Nevertheless, the responses were proportional to their predicted values (.363 vs. 340). The advantage of WLS was due to the mechanics of selection resulting in slightly greater selection intensity and an increased correlation between the criterion of selection and the trait being improved. On the contrary, the RRS method had a slightly larger realized heritability as would be expected in the presence of non-additive genetic variance. Four of five pure-lines selected under both methods had statistically significant declines in performance due to inbreeding depression effects. All of the results observed are comparable with known genetic theory.Some problems in comparing genetic gains from different selection methods or selection criteria are discussed.
RESUMO
Pregnant rats were injected with vehicle or 1,2 microgram diethylstilbestrol (DES) during wk 2 or 3 of gestation; their female offspring ( approximately 50 d old) were fed 7,12-dimethylbenz[a]anthrocene (DMBA). The survivors (27 per group) were sacrificed 30 wk later. The three groups did not differ in the number of tumor-bearing animals; however, significantly more palpable mammary tumors arose in both DES-exposed groups than in controls. When DES was given during the second trimester, palpable tumors appeared earlier than in the other two groups. Thus, transplancental exposure to DES potentiated the action of a known carcinogen (DMBA) on rat mammary tissue. These results raise the possibility that, for young women, DES exposure in utero may have affected tissues other than the vagina. Further investigation is warranted, with special emphasis on the effects of DES on mammary and other estrogen-sensitive tissues.
Assuntos
9,10-Dimetil-1,2-benzantraceno/farmacologia , Benzo(a)Antracenos/farmacologia , Dietilestilbestrol/toxicidade , Neoplasias Mamárias Experimentais/induzido quimicamente , Animais , Feminino , Troca Materno-Fetal , Gravidez , Ratos , Fatores de TempoRESUMO
The stoichiometry of the reaction between [14C]-9-cis-retinoyl fluoride, a close isostere of 9-cis-retinal, and bovine opsin and the biochemical and spectral properties of this new pigment were investigated. The stoichiometry of retinoid incorporation is approximately one in dodecyl maltoside, a detergent in which opsin is capable of regeneration with 11-cis-retinal. Interestingly, in Ammonyx LO, a detergent that does not permit rhodopsin regeneration, the stoichiometry of binding is still approximately one. By contrast, heat-denatured opsin does not irreversibly bind substantial [14C]retinoyl fluoride. This result strongly suggests that the nucleophilicity of the active site lysine is retained in Ammonyx LO but that further conformational changes in the protein, required to form rhodopsin, are not possible. These results are all consistent with an active site directed mechanism for the irreversible reaction of 9-cis-retinoyl fluoride with opsin probably at the active site lysine residue. The ultraviolet spectra of 9-cis-retinoylopsin and its all-trans congener show gamma max's at 373 and 380 nm, respectively, somewhat bathochromically shifted from their respective model N-butylretinamides which absorb at 347 and 351 nm. Photolysis of both 9-cis- and all-trans-retinoylopsins leads to the same photostationary state. This shows that, as expected, photoisomerization without bleaching occurs. The photolysis of either 9-cis- or all-trans-retinoylopsin in the presence of the G protein (transducin) does not lead to the activation of the latter. This is consistent with the notion that a protonated Schiff base is critical for the function of rhodopsin.
Assuntos
Proteínas do Olho/metabolismo , Células Fotorreceptoras/metabolismo , Pigmentos da Retina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Tretinoína/análogos & derivados , Animais , Radioisótopos de Carbono , Bovinos , GTP Fosfo-Hidrolases/metabolismo , Cinética , Radioisótopos de Fósforo , Opsinas de Bastonetes , Estereoisomerismo , Tretinoína/metabolismoRESUMO
The absorption of a photon of light by rhodopsin results in the cis to trans isomerization of the 11-cis-retinal Schiff base chromophore. In the studies reported here, an attempt is made to determine the mechanism of the energization of rhodopsin as it relates to the chemistry of the isomerization process and the geometrical state of the chromophore. Studies were performed with vitamin A analogues to probe this mechanism. Both 11-cis-7,8-dihydroretinal and 9-cis-7,8-dihydroretinal form bleachable pigments when combined with opsin. Photolysis of these pigments in the presence of G-protein results in the activation of the latter as revealed by its GTPase activity. Phosphodiesterase is also activated when it is included in the incubation. Therefore, the possibility that rhodopsin is energized by mechanisms involving photochemically induced charge transfer from the protonated Schiff base to the beta-ionone ring can be discarded. Further studies were conducted with all-trans-vitamin A derivatives to determine if these compounds can form the GTPase-activating state R*, a situation that is possible, in principle, by microscopic reversibility. Neither all-trans-retinal nor its oxime, when incubated with bovine opsin in the dark, caused activation of the GTPase, requiring at least a 5 kcal/mol energy gap between them. Furthermore, stoichiometric adducts of all-trans-retinoids and opsin were also unable to mediate activation of the GTPase. Since both all-trans-15,16-dihydroretinylopsin and all-trans-retinoylopsin possess an all-trans-retinoid permanently adducted to opsin, it can be concluded that the all-trans-retinoid chromophore-opsin linkage may be necessary but not sufficient to achieve activation of the visual pigment.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Pigmentos da Retina/metabolismo , Retinoides/metabolismo , Rodopsina/metabolismo , Animais , Bovinos , GMP Cíclico/metabolismo , Ativação Enzimática , Guanosina Monofosfato/metabolismo , Guanosina Trifosfato/metabolismo , Isomerismo , Ligação Proteica , Segmento Externo da Célula Bastonete/metabolismo , Relação Estrutura-AtividadeRESUMO
Aspects of development and morphology were studied in the reproductive tract of female ACI rats exposed prenatally to diethylstilbestrol (DES) and followed to 10 months of age. Pregnant ACI rats were injected with vehicle or DES (0.8 microgram = low DES or 8.0 micrograms = high DES) on Days 15 and 18 of gestation. At 12 weeks of age, half of the female offspring in each prenatal exposure group received a subcutaneous implant of a pellet containing 2.5 mg DES and 17.5 mg cholesterol; the remaining offspring received a control cholesterol pellet. Maternal reproductive performance was significantly impaired in DES-treated dams compared to controls. In female offspring mean time of vaginal opening was accelerated from 50.3 +/- 2.7 days in the vehicle-exposed group to 46.2 +/- 2.6 and 47.1 +/- 2.3 days in the low and high DES groups, respectively. Prior to pellet implantation, none of the rats exposed to DES prenatally was in "persistent estrus." At necropsy, rats exposed to DES in utero and implanted with the cholesterol pellet showed an increased frequency of atypical uterine epithelia, cystically dilated uterine glands, and a thickened vaginal epithelium. Among groups implanted with the DES pellet, prenatal exposure to DES increased the incidence of squamous metaplasia of the luminal epithelium and of cystically dilated uterine glands. Collectively, groups implanted with the DES pellet had higher incidences of squamous metaplasia of the uterine lumen, cystically dilated uterine glands, and patches of multilayered uterine epithelium than groups bearing the cholesterol pellet. DES pellet-bearing rats were also found to display a pronounced thickening and vacuolation of the vaginal epithelium. Cervical tissue from 98% of the DES-treated litters was characterized by a markedly convoluted epithelium with numerous squamous cell nests. There were no apparent effects of prenatal DES exposure or postnatal DES treatment on ovarian or oviductal histology. However, ovarian wet weights were significantly reduced as a result of postnatal DES treatment. Thus, the epithelial tissues of the uterus, cervix, and vagina in the ACI rat show a sensitivity to DES whether administered prenatally, postnatally, or in combination.
Assuntos
Dietilestilbestrol/toxicidade , Genitália Feminina/patologia , Animais , Implantes de Medicamento , Células Epiteliais , Epitélio/efeitos dos fármacos , Tubas Uterinas/ultraestrutura , Feminino , Genitália Feminina/efeitos dos fármacos , Troca Materno-Fetal , Ovário/patologia , Gravidez , Ratos , Ratos Endogâmicos ACI , Valores de Referência , Útero/patologia , Vagina/patologiaRESUMO
Photolysis of rhodopsin leads to the formation of an activated intermediate that activates a G protein, thus beginning the visual cascade. This activated form of rhodopsin appears coincident in time with the spectroscopically defined intermediate, metarhodopsin II. Metarhodopsin I, the precursor of metarhodopsin II, contains a protonated Schiff base, whereas metarhodopsin II does not. The question of whether the deprotonation of the protonated Schiff base is obligate in the formation of activated rhodopsin was addressed by monomethylating the active-site lysine of permethylated rhodopsin and determining whether this pigment can activate the G protein upon photolysis. The photolysis of the new pigment, which absorbs at 520 nm, led to the formation of a relatively stable metarhodopsin I-like intermediate with a lambda max of approximately equal to 485 nm, with no apparent formation of either metarhodopsin II- or metarhodopsin III-like intermediates. The only probe available to detect formation of the active form of rhodopsin is G protein activation. Photolysis of the pigment in the presence of the G protein did not lead to measurable activation of the GTPase activity of the latter. These studies establish a functional link between Schiff base deprotonation and activation of the G protein. It is concluded that proton transfer from the protonated Schiff base of rhodopsin is obligate for the initiation of visual transduction.
Assuntos
GTP Fosfo-Hidrolases , Proteínas de Ligação ao GTP , Monoéster Fosfórico Hidrolases , Pigmentos da Retina , Rodopsina , Visão Ocular , Animais , Sítios de Ligação , Bovinos , Ativação Enzimática , Técnicas In Vitro , Metilação , Conformação Proteica , Prótons , Retina/fisiologia , Bases de Schiff , Análise Espectral , Relação Estrutura-AtividadeRESUMO
The studies reported are concerned with the functional consequences of the chemical modifications of the lysines and carboxyl-containing amino acids of bovine rhodopsin. The 10 non-active-site lysine residues of rhodopsin can be completely dimethylated and partially acetimidated (8-9 residues) with no loss in the ability of the proteins to activate the G protein when photolyzed or to regenerate with 11-cis-retinal. These modifications do not alter the net charge on the protein. Surprisingly, heavy acetylation of these lysines (eight to nine residues) with acetic anhydride, which neutralizes the positive charges of the lysine residues, yields a modified rhodopsin fully capable of activating the G protein and being regenerated. It is concluded that the non-active-site lysine residues of rhodopsin are not importantly and directly involved in interactions with the G protein during photolysis. However, this is not to say that they are unimportant in maintaining the tertiary structure of the protein because heavy modification of these residues by succinylation and trinitrophenylation produces proteins incapable of G protein activation, although the succinylated protein still regenerated. The active-site lysine of rhodopsin was readily modified and prevented from regenerating with 11-cis-retinal and with o-salicylaldehyde and o-phthalaldehyde/mercaptoethanol, two sterically similar aromatic aldehyde containing reagents which react by entirely different mechanisms. It is suggested that rhodopsin contains an aromatic binding site within its active-site region. Monoethylation, but not monomethylation, of the active-site lysine also prevented regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas do Olho/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Células Fotorreceptoras/metabolismo , Pigmentos da Retina/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Animais , Radioisótopos de Carbono , Bovinos , Formaldeído/metabolismo , Cinética , TransducinaRESUMO
Cyclic nucleotide stimulated efflux of 22Na+ and 45Ca2+ from a purified bovine rod outer segment disk preparation was measured on the 25-100-ms time scale by a novel rapid superfusion method. Activation of cation efflux by 8-bromoguanosine cyclic 3',5'-phosphate (8-Br-cGMP) was maximal within 25 ms. Over a wide range of concentrations of 8-Br-cGMP, the kinetics of termination of efflux precisely conformed to the sum of two exponential decay processes: a rapid phase (decay constant of 200 ms) and a slower phase (decay constant of 1.6 s). The kinetics of the biphasic decay of efflux cannot be explained by depletion of a pool of releasable 22Na but appear to reflect an intrinsic process for inactivation of the channels. 8-Br-cGMP-stimulated release of actively accumulated 45Ca exhibited identical biphasic decay kinetics. The maximum rate of Ca release [5 nmol.(mg of disk protein)-1.min-1] may be sufficient to produce a 1 microM change in local cytoplasmic [Ca] within 20 ms. The Ca:Na selectivity ratio is approximately 0.5:1 for both decay phases. 8-Br-cGMP demonstrated a lower potency (EC50 of 8.4 microM vs 2.8 microM) but a higher degree of cooperativity in its activation of the rapid vs the slower decay phase of 22Na efflux. The slower phase of decay was selectively inhibited by 25 microM l-cis-diltiazem, a relatively weak inhibitor of the rapid decay phase. Sodium ion (5-10 mM) selectively inhibited the rapid decay phase of 8-Br-cGMP-stimulated 45Ca release. These two kinetically and pharmacologically distinct phases of decay are hypothesized to represent two functionally distinct forms of cGMP-stimulated cation channels.
Assuntos
GMP Cíclico/farmacologia , Canais Iônicos/efeitos dos fármacos , Células Fotorreceptoras/efeitos dos fármacos , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Amilorida/farmacologia , Animais , Cálcio/metabolismo , Cátions Monovalentes , Bovinos , Cinética , Segmento Externo da Célula Bastonete/metabolismo , Sódio/metabolismo , EstereoisomerismoRESUMO
Various characteristics of steroid binding proteins from mammary tumors and uteri of rats exposed prenatally to diethylstilbestrol (DES) were examined. Pregnant rats were treated with no hormone (group A) or with a total dose of 1.2 micrograms DES during the second (group B) or third (group C) trimester of gestation. Female offspring received 7,12-dimethylbenz[a]anthracene (DMBA) at d 50 +/- 1. Animals with large mammary tumors were subjected to bilateral ovariectomy. Seven months after carcinogen treatment, the experiment was terminated. High-affinity binding sites for [3H] estradiol-17 beta and [3H]R5020 were found in all mammary tumors assayed. On sucrose gradients of low ionic strength both 8S and 4S forms of the estrogen receptor were identified in mammary tumors, regardless of prenatal treatment. In addition, progestin receptors sedimenting at 4S were identified in these tumors. However, the 7-8S form of the progestin receptor was found only in tumors from intact animals. Levels of progestin receptors were diminished after ovariectomy, both in mammary tumors and in uteri; ovariectomy also resulted in a significant reduction in uterine wet weight in the hormone exposure groups, as expected. Unlike groups A and B, rats exposed to DES during the third trimester had uterine progestin binding capacities and uterine wet weights that did not decrease proportionally ater ovariectomy. Furthermore, progestin binding capacities in mammary tumors from group C ovariectomized rats were higher than those in the other two groups. In intact rats from group C, cytosol from mammary tumors also had elevated levels of progestin binding; however, no differences in progestin binding were observed in the uteri from these animals. Small differences in estrogen binding capacities in tumor tissues were observed among the three groups; uterine estrogen binding capacities did not vary significantly. Prenatal exposure to DES during the third trimester appeared related to persistent biochemical alterations in rat mammary tumors and uteri; earlier exposure did not have this effect.
Assuntos
9,10-Dimetil-1,2-benzantraceno , Benzo(a)Antracenos , Dietilestilbestrol/toxicidade , Neoplasias Mamárias Experimentais/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Útero/análise , Animais , Castração , Estradiol/metabolismo , Feminino , Neoplasias Mamárias Experimentais/análise , Gravidez , Promegestona/metabolismo , Ratos , Ratos Endogâmicos , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.