RESUMEN
Anabolic ornithine transcarbamoylase (aOTC) catalyzes the reaction between carbamoyl phosphate (CP) and L-ornithine (ORN) to form L-citrulline and phosphate in the urea cycle and L-arginine biosynthesis. The crystal structure of unliganded aOTC from Campylobacter jejuni (Cje aOTC) was determined at 2.7 Å resolution and refined to an R(work) of 20.3% and an R(free) of 24.0%. Cje aOTC is a trimer that forms a head-to-head pseudohexamer in the asymmetric unit. Each monomer is composed of an N-terminal CP-binding domain and a C-terminal ORN-binding domain joined by two interdomain helices. The Cje aOTC structure presents an open conformation of the enzyme with a relatively flexible orientation of the ORN-binding domain respective to the CP-binding domain. The conformation of the B2-H3 loop (residues 68-78), which is involved in binding CP in an adjacent subunit of the trimer, differs from that seen in homologous proteins with CP bound. The loop containing the ORN-binding motif (DxxxSMG, residues 223-230) has a conformation that is different from those observed in unliganded OTC structures from other species, but is similar to those in structures with bound ORN analogs. The major differences in tertiary structure between Cje aOTC and human aOTC are described.
Asunto(s)
Campylobacter jejuni/enzimología , Ornitina Carbamoiltransferasa/química , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
In the mammalian ovary both gonadotropins and local cytokines, acting through G-protein coupled receptors, govern the physiology of the ovary in part by regulating the cyclic adenosine 3',5'-monophosphate via adenylyl cyclases. The nine transmembrane adenylyl cyclases and a soluble adenylyl cyclase are regulated by a diversity of ligands. In this study we have examined the rat ovaries, prior to and subsequent to gonadotropin treatment, for the presence of different transmembrane adenylyl cyclases by indirect immunofluorescence microscopy. Adenylyl cyclase I immunoreactivity was observed in the nuclei of oocytes in preantral and antral follicles along with some staining in granulosa cells. Equine chorionic gonadotropin injection increased adenylyl cyclase I staining in granulosa cells. Adenylyl cyclase I staining was also observed in luteal and endothelial cells. Adenylyl cyclase II was observed throughout the ovary, including granulosa cells and the ovarian surface epithelium. Adenylyl cyclase II staining was also found to increase in granulosa cells after equine chorionic gonadotropin injection. Adenylyl cyclase III was distributed primarily in theca and smooth muscle cells of arterioles, with faint staining in the oocytes of equine chorionic gonadotropin-injected ovaries. Adenylyl cyclase IV staining was present throughout the ovary, including the nuclei of oocytes. Adenylyl cyclase VIII staining in granulosa cells increased subsequent to equine chorionic gonadotropin injection and remained in luteal cells. Our study reveals the redundancy of adenylyl cyclases present in the rat ovary and, therefore, implies potential regulation of follicular and corpus luteum physiology by cyclic adenosine 3',5'-monophosphate generated through distinct adenylyl cyclases.
Asunto(s)
Adenilil Ciclasas/metabolismo , Ovario/enzimología , Animales , Femenino , Humanos , Microscopía Fluorescente/métodos , RatasRESUMEN
Although the effects of Delta(9)-tetrahydrocannabinol (THC) on ovarian physiology have been known for many decades, its mechanism of action in the rat ovary remains poorly understood. The effects of THC and endocannabinoids on many cell types appear to be mediated through the G-protein-coupled CB(1) and CB(2) receptors. Evidence also suggests that the concentration of the endocannabinoid anandamide is regulated by cellular fatty acid amide hydrolase (FAAH). Therefore, we examined the rat ovary for the presence of CB(1) and CB(2) receptors and FAAH. The CB(1) receptor was present in the ovarian surface epithelium (OSE), the granulosa cells of antral follicles, and the luteal cells of functional corpus luteum (CL). The granulosa cells of small preantral follicles, however, did not express the CB(1) receptor. Western analysis also demonstrated the presence of a CB(1) receptor. In both preantral and antral follicles, the CB(2) receptor was detected only in the oocytes. In the functional CL, the CB(2) receptor was detected in the luteal cells. FAAH was codistributed with CB(2) receptor in both oocytes and luteal cells. FAAH was also present in the OSE, subepithelial cords of the tunica albuginea (TA) below the OSE, and in cells adjacent to developing preantral follicles. Western analysis also demonstrated the presence of FAAH in oocytes of both preantral and antral follicles. Our observations provide potential explanation for the effects of THC on steroidogenesis in the rat ovary observed by earlier investigators and a role for FAAH in the regulation of ovarian anandamide.
Asunto(s)
Amidohidrolasas/metabolismo , Ovario/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Animales , Ácidos Araquidónicos/metabolismo , Western Blotting , Moduladores de Receptores de Cannabinoides/metabolismo , Cuerpo Lúteo/metabolismo , Dronabinol/farmacología , Endocannabinoides , Femenino , Células de la Granulosa/metabolismo , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Ovario/efectos de los fármacos , Alcamidas Poliinsaturadas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Changes in metabolic state, such as those induced by fasting, have profound effects on reproduction. In rats, the time-course over which fasting inhibits luteinising hormone (LH) release is reduced to 48 h by the presence of oestradiol-17beta (E(2)). Hypothalamic kisspeptin plays a key role in mediating the actions of E(2) on gonadotrophin-releasing hormone (GnRH) neurones, and thereby promotes LH release. KiSS-1-expressing neurones are found in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). Extensive evidence implicates the AVPV in GnRH release and the ARC in energy balance. The latter nucleus also contains neurones that express neuropeptide Y (NPY), an orexigenic peptide implicated in GnRH control. To elucidate the involvement of kisspeptin and/or NPY in hypothalamic responses to fasting, their expression was quantified by in situ hybridisation histochemistry in ovariectomised rats, with or without E(2) replacement, before and after 48 h of fasting. In the presence of E(2), but not in its absence, the fasting suppressed plasma LH. In the AVPV, the low level of KiSS-1 expression found in the absence of E(2) was unaffected by fasting. By contrast, the elevated level found in the presence of E(2) was suppressed by fasting. Independent of E(2), fasting had no effect on KiSS-1 expression in the ARC, but increased NPY expression at that site. The present study has identified the AVPV as a site at which KiSS-1 expression can be influenced by fasting. The results suggest that inhibition of KiSS-1 expression in the AVPV may be a significant factor in restraining the gonadotrophic axis in response to negative energy balance in the presence of oestrogen. The extent to which the concurrent rise in NPY expression in the ARC may contribute to the suppression of LH release by influencing AVPV kisspeptin neurones, directly or indirectly, or by actions independent of kisspeptin, remains to be established.
Asunto(s)
Núcleos Talámicos Anteriores/metabolismo , Núcleo Arqueado del Hipotálamo/metabolismo , Ayuno/fisiología , Neuropéptido Y/genética , Proteínas/genética , Animales , Peso Corporal/fisiología , Regulación hacia Abajo/genética , Ayuno/sangre , Ayuno/metabolismo , Femenino , Kisspeptinas , Hormona Luteinizante/sangre , Neuronas Aferentes/metabolismo , Neuropéptido Y/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
Orexin A stimulates GnRH release from hypothalamic explants in vitro. The sites of action of orexin A in the regulation of LH release have been investigated in vivo in ovariectomized rats that were given vehicle or estradiol benzoate (EB), with or without an injection of progesterone 48 h later. Orexin A was administered intrahypothalamically under Saffan anesthesia, 50 h after the EB or vehicle; its effects on plasma LH levels were monitored in sequential blood samples. Orexin A (1.0 microg/side) injected into the rostral preoptic area (rPOA) at the level of the organum vasculosum of the lamina terminalis had a stimulatory effect on LH release in EB-treated ovariectomized rats. When orexin A was injected into the medial POA (mPOA) or the arcuate/median eminence, it had an inhibitory effect on the LH surge that occurs in ovariectomized rats primed with EB plus progesterone. Orexin A injected into the mPOA also reduced LH levels in ovariectomized rats untreated with ovarian steroids. Both the stimulatory and inhibitory effects of orexin A were antagonized by SB334867A, a selective orexin 1 receptor antagonist. Furthermore, when given alone into the rPOA, this antagonist attenuated the LH surge induced by EB plus progesterone. Thus, orexin appears to have a dual effect on LH release, being stimulatory in the rPOA and inhibitory in the mPOA or arcuate/median eminence. Both effects may be mediated, at least in part, by the orexin 1 receptor. Double label immunohistochemistry revealed close appositions between orexin A immunoreactive varicosities and a small proportion of GnRH cell bodies in the rPOA. It is suggested that the stimulatory effect of orexin A on LH release may involve direct actions on GnRH neurons.
Asunto(s)
Proteínas Portadoras/farmacología , Estradiol/análogos & derivados , Péptidos y Proteínas de Señalización Intracelular , Hormona Luteinizante/metabolismo , Neuropéptidos/farmacología , Área Preóptica/efectos de los fármacos , Área Preóptica/metabolismo , Urea/análogos & derivados , Animales , Benzoxazoles/farmacología , Proteínas Portadoras/metabolismo , Células Cultivadas , Estradiol/farmacología , Femenino , Hormona Liberadora de Gonadotropina/metabolismo , Inmunohistoquímica , Microinyecciones , Naftiridinas , Neuropéptidos/metabolismo , Receptores de Orexina , Orexinas , Ovariectomía , Área Preóptica/citología , Progesterona/farmacología , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G , Receptores de Neuropéptido/antagonistas & inhibidores , Urea/farmacologíaRESUMEN
It is widely accepted that axillary malodour is attributable to the microbial biotransformation of odourless, natural secretions into volatile odorous products. Consequently, there is a need to understand the microbial ecology of the axilla in order that deodorant products, which control microbial action in this region, can be developed in the appropriate manner. A detailed characterization of the axillary microflora of a group of human volunteers has been performed. The axillary microflora is composed of four principal groups of bacteria (staphylococci, aerobic coryneforms, micrococci and propionibacteria), and the yeast genus Malassezia. Results indicated that the axillary microflora was dominated by either staphylococcal or aerobic coryneform species. Comparisons between axillary bacterial numbers and levels of axillary odour demonstrated the greatest association between odour levels and the presence of aerobic coryneforms in the under-arm. As the taxonomy of cutaneous aerobic coryneforms is poorly understood, a further study was conducted to characterize selected axillary aerobic coryneform isolates. Using the molecular technique of 16S rDNA sequencing, selected genomic sequences of a number of axillary aerobic coryneform isolates were obtained. Comparisons with sequence databases indicated the likely presence of a range of Corynebacterium species on axillary skin, although the majority of isolates were most similar to either Corynebacterium G-2 CDC G5840 or C. mucifaciens DMMZ 2278. Although for a panel of individuals differences in the carriage of Corynebacterium species were noted, similar species were carried by a number of panellists. All isolates examined in this limited evaluation failed to demonstrate the capability to metabolize long-chain fatty acids (LCFAs) to shorter chain, more volatile products. The application of this modern molecular phylogenetic technique has increased understanding of the diversity of aerobic coryneform carriage in the axilla, and on human skin. The application of this technique in other studies to assess the ethnic differences in cutaneous bacterial ecology, or the effects on the microflora of specific product use, will assist in the future development of novel deodorant systems.
RESUMEN
It is proposed that the orientation of elongate objects, such as bones, may be used to identify the flow direction of ancient river deposits. If true, elongate objects could be of great value when ancient bedforms such as ripples and dunes are not visible. Two sandstone quarries were investigated wherein the paleoflow direction was determined from both bedforms and elongate dinosaur bones. A mixture of two von Mises distributions captures the observation that elongate bones transported under unidirectional flow conditions will align both parallel and perpendicular to the flow direction. Likelihood ratio tests for a mixture of two von Mises distributions are given. The power of these tests is investigated by simulation since the direction of dinosaur bones agrees with the primary bedforms if the hypothesis test comparing the dominant mean direction of the bones to the paleoflow direction fails to reject. The likelihood ratio test on the dominant mean direction has reasonable power. If the two mean directions in the mixture distribution are pi apart, a more powerful likelihood ratio test can be used. The likelihood ratio test on the hypothesis that the two mean directions are exactly pi apart is useful in determining if the assumptions of the more powerful test are satisfied.
Asunto(s)
Biometría , Funciones de Verosimilitud , Animales , Simulación por Computador , Fósiles , Fenómenos Geológicos , Geología , Modelos Estadísticos , Paleontología/estadística & datos numéricos , ReptilesRESUMEN
A high resolution NMR structure of hen lysozyme has been determined using 209 residual 1H-15N dipolar coupling restraints from measurements made in two different dilute liquid crystalline phases (bicelles) in conjunction with a data set of 1632 NOE distance restraints, 110 torsion angle restraints, and 60 hydrogen bond restraints. The ensemble of 50 low-energy calculated structures has an average backbone RMSD of 0.50+/-0.13A to the mean structure and of 1.49+/-0.10A to the crystal structure of hen lysozyme. To assess the importance of the dipolar coupling data in the structure determination, the final structures are compared with an ensemble calculated using an identical protocol but excluding the dipolar coupling restraints. The comparison shows that structures calculated with the dipolar coupling data are more similar to the crystal structure than those calculated without, and have better stereochemical quality. The structures also show improved quality factors when compared with additional dipolar coupling data that were not included in the structure calculations, with orientation-dependent 15N chemical shift changes measured in the bicelle solutions, and with T1/T2 values obtained from 15N relaxation measurements. Analysis of the ensemble of NMR structures and comparisons with crystal structures, 15N relaxation data, and molecular dynamics simulations of hen lysozyme provides a detailed description of the solution structure of this protein and insights into its dynamical behavior.
Asunto(s)
Estructura Molecular , Muramidasa/química , Resonancia Magnética Nuclear Biomolecular/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Animales , Aspergillus niger/química , Aspergillus niger/genética , Pollos , Cristalografía por Rayos X , Femenino , Técnicas In Vitro , Conformación Molecular , Conformación ProteicaRESUMEN
Pulse field gradient NMR methods have been used to determine the effective hydrodynamic radii of a range of native and nonnative protein conformations. From these experimental data, empirical relationships between the measured hydrodynamic radius (R(h)) and the number of residues in the polypeptide chain (N) have been established; for native folded proteins R(h) = 4.75N (0.29)A and for highly denatured states R(h) = 2.21N (0.57)A. Predictions from these equations agree well with experimental data from dynamic light scattering and small-angle X-ray or neutron scattering studies reported in the literature for proteins ranging in size from 58 to 760 amino acid residues. The predicted values of the hydrodynamic radii provide a framework that can be used to analyze the conformational properties of a range of nonnative states of proteins. Several examples are given here to illustrate this approach including data for partially structured molten globule states and for proteins that are unfolded but biologically active under physiological conditions. These reveal evidence for significant coupling between local and global features of the conformational ensembles adopted in such states. In particular, the effective dimensions of the polypeptide chain are found to depend significantly on the level of persistence of regions of secondary structure or features such as hydrophobic clusters within a conformational ensemble.
Asunto(s)
Adhesinas Bacterianas , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas/química , Animales , Aprotinina/química , Proteínas Bacterianas/química , Proteínas Portadoras/química , Grupo Citocromo c/química , Muramidasa/química , Mioglobina/química , Péptidos/química , Fosfatidilinositol 3-Quinasas/química , Desnaturalización Proteica , Estreptoquinasa/química , Termodinámica , Triosa-Fosfato Isomerasa/químicaRESUMEN
Oxidized and reduced hen lysozyme denatured in 8 M urea at low pH have been studied in detail by NMR methods. 15N correlated NOESY and TOCSY experiments have provided near complete sequential assignment for both 1H and 15N resonances. Over 900 NOEs, including 130 (i, i + 2) and 23 (i, i + 3) NOEs, could be identified by analysis of the NOESY spectra of the denatured states, and 3J(HN, Halpha) coupling constants and 15N relaxation rates have been measured. The coupling constant and NOE data were analyzed by comparisons with theoretical predictions from a random coil polypeptide model based on amino acid specific phi,psi distributions extracted from the protein data bank. There is significant agreement between predicted and experimental NMR parameters suggesting that local conformations of the denatured states are largely determined by short-range interactions within the polypeptide chain. This result is supported by the observation that the chemical shift, coupling constant, and NOE data are little affected by whether or not the four disulfide bridge cross-links are formed in the denatured protein. The relaxation data, however, show significant differences between the oxidized and reduced protein. Analysis of the relaxation data in terms of simple dynamics models provides evidence for weak clustering of hydrophobic groups near tryptophan residues and increased barriers to motion in the more compact conformers formed when the polypeptide chain is cross-linked by the disulfide bridges. Using this information, a structural description of these denatured states is given in terms of an ensemble of conformers, which have a complex relationship between their local and global characteristics.
Asunto(s)
Modelos Químicos , Muramidasa/química , Secuencia de Aminoácidos , Animales , Aspergillus niger , Pollos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Método de Montecarlo , Conformación Proteica , Desnaturalización Proteica , UreaRESUMEN
The effects of the commonly used denaturant guanidine hydrochloride(GuHCl) on the random coil conformations and NMR chemical shifts of theproteogenic amino acids have been characterized using the peptide seriesAc-Gly-Gly-X-Gly-Gly-NH(2). The phi angle-sensitive couplingconstants, ROESY cross peak intensities and proline cis-trans isomerratios of a representative subset of these peptides are unaffected by GuHCl,which suggests that the denaturant does not significantly perturb intrinsicbackbone conformational preferences. A set of(3)J(HNHalpha) values is presented which agreewell with predictions of recently developed models of the random coil. Wehave also measured the chemical shifts of all 20 proteogenic amino acids inthese peptides over a range of GuHCl concentrations. The shifts exhibit alinear dependence on denaturant concentration and we report here correctionfactors for the calculation of 'random coil' (1)H chemicalshifts at any arbitrary denaturant concentration. Studies of arepresentative subset of peptides indicate that (13)C and(15)N chemical shifts are also perturbed by the denaturant.These results should facilitate the application of chemical shift-basedanalytical techniques to the study of polypeptides in solution with GuHCl.The effects of the denaturant on the quality of NMR spectra and on chemicalshift referencing are also addressed.
RESUMEN
Primary cutaneous Bacillus cereus infection frequently presents as a single necrotic bulla on the extremity of an immunocompromised patient. In lesional biopsy specimens and smears, the large gram-positive rods of B cereus may be mistaken for Clostridium species. This is a potentially serious error, as Bacillus species are resistant to penicillin and other beta-lactam antibiotics. We studied a case in which large periodic acid-Schiff-staining organisms were seen in the biopsy specimen from a necrotic bulla on the finger of a neutropenic patient with diffuse large cell lymphoma. The tissue biopsy specimen subsequently yielded a pure culture of B cereus. Staining with periodic acid-Schiff was then performed on a series of bacterial species in human tissue and from smears of culture colonies. The following bacterial species were found to be consistently periodic acid-Schiff positive after diastase digestion: B cereus, Corynebacterium diphtheriae, Propionibacterium acnes, Klebsiella pneumoniae, and Micrococcus luteus.
Asunto(s)
Bacillus cereus/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Reacción del Ácido Peryódico de Schiff , Enfermedades Cutáneas Infecciosas/diagnóstico , Anciano , Bacterias/aislamiento & purificación , Diagnóstico Diferencial , Humanos , MasculinoRESUMEN
Among Inuit less than 30 years old the prevalence of myopia is far in excess of that of their elders. This is especially true for females. There seems to be little, if any, genetic contribution to this "epidemic" of myopia in the young. The age and sex distribution indicates the likelihood of an environmental factor, probably cultural, being responsible for the current pattern. Other data implicate school attendance as a possible etiologic factor.