RESUMEN
In most yeast species, the mitochondrial DNA (mtDNA) has been reported to be a circular molecule. However, two cases of linear mtDNA with specific termini have previously been described. We examined the frequency of occurrence of linear forms of mtDNA among yeasts by pulsed-field gel electrophoresis. Among the 58 species from the genera Pichia and Williopsis that we examined, linear mtDNA was found with unexpectedly high frequency. Thirteen species contained a linear mtDNA, as confirmed by restriction mapping, and labeling, and electron microscopy. The mtDNAs from Pichia pijperi, Williopsis mrakii, and P. jadinii were studied in detail. In each case, the left and right terminal fragments shared homologous sequences. Between the terminal repeats, the order of mitochondrial genes was the same in all of the linear mtDNAs examined, despite a large variation of the genome size. This constancy of gene order is in contrast with the great variation of gene arrangement in circular mitochondrial genomes of yeasts. The coding sequences determined on several genes were highly homologous to those of the circular mtDNAs, suggesting that these two forms of mtDNA are not of distant origins.
Asunto(s)
ADN de Hongos/química , ADN Mitocondrial/química , Levaduras/genética , Secuencia de Bases , Clasificación , Electroforesis en Gel de Campo Pulsado , Genes Fúngicos , Ligamiento Genético , Microscopía Electrónica , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
In some strains of Saccharomyces cerevisiae, the induction of enzymes of the Leloir pathway, galactose fermentation and growth on galactose depend on mitochondrial function; mitochondrial dependence is elicited through the recessive allele imp1 of the nuclear gene IMP1. The genetic element IMP1 is not allelic to any of the known GAL genes; IMP1 strains can grow on and ferment galactose in respiratory-deficient (RD) condition or in the presence of the mitochondrial inhibitors ethidium bromide and erythromycin; whereas, imp1 strains can grow on and ferment galactose only in respiratory-sufficient (RS) condition. The imp1 elicited mitochondrial dependence apparently involves regulation of the synthesis of the galactose catabolizing enzymes and synthesis of the galactose specific permease. IMP1 is not the only genetic determinant that elicits an interaction of the mitochondrion and the expression of the Gal system; the GAL3 gene, whose role in galactose utilization is demonstrated by the long-term adaptation phenotype of gal3 rS mutants, gives rise to a noninducible phenotype in RD condition or in the presence of mitochondrial inhibitors.
Asunto(s)
ADN de Hongos/genética , ADN Mitocondrial/genética , Galactosa/metabolismo , Saccharomyces cerevisiae/genética , Núcleo Celular/fisiología , Inducción Enzimática , Fermentación , Genes Recesivos , Fenotipo , Saccharomyces cerevisiae/metabolismoRESUMEN
RESUMEN Diversos estudios bioquímicos adicionales a la evaluación de Testosterona total (TT), biodisponible (Tbio) y libre (TL) han sido realizados a los efectos que pudieran resultar de mayor utilidad para el diagnóstico de patologías concomitantes en el SOP, entre otros. En la hormona anti Mülleriana, cuando la concentración supera a los 3,0 ng/ml existen evidencias de que el 79% de las mismas pueden ser identificadas correctamente como SOP. El Antígeno Prostático Específico (PSA), marcador de singular importancia en pacientes con cáncer de Próstata, con técnicas ultrasensibles ha podido ser detectado en más del 50% en mujeres. En un grupo de pacientes con SOP, los niveles circulantes de PSA fueron significativamente mayores que en las mujeres sin SOP. El Kiss-1 aislado de la placenta y demostrado en otros tejidos, presenta niveles aumentados que correlacionan con la LH, TT, TL y resistencia a la insulina (RI) en adolescentes con SOP versus adolescentes sin SOP, sugiriendo que el Kiss-1 podría estar involucrado en el desarrollo del SOP en estas pacientes. Algunas pacientes con SOP están asociadas a patologías relevantes, de las cuales han sido comunicadas el aumento del BMI, mayor grado de dislipemia, adiposidad central, RI y Síndrome Metabólico (SMe). En las pacientes con un fenotipo clásico (hiperandrogenismo, alteración del ciclo menstrual y ovarios poliquísticos), estas patologías son de mayor frecuencia y severidad que en los otros fenotipos, particularmente aquellos sin hiperandrogenismo. Otras determinaciones como TNFα, interleuquinas, test de tolerancia a la glucosa, ApoB, partículas pequeñas de LDL e Inhibidor del Activador del Plasminógeno-1 han sido comunicados que podrían ser de utilidad para tener mayor sensibilidad en la definición de patología concomitantes en el SOP. Actualmente se ha comenzado a evaluar otros marcadores como el Fetuin-A; Quemerina, Nesfatina-1, Neopterina y Endocannabinoides, cuyos resultados preliminares parecerían ser un aporte importante para evaluar SMe y RI en paciente con SOP y tratar de definir su prevalencia en los distintos fenotipos de esta patología.
ABSTRACT Several biochemical studies in addition to the evaluation of total Testosterone (TT), bioavailable (bioT) and free (FT) have been performed to the effects that could be of greater use for the diagnosis of concomitant pathologies in the PCOS, among others. The anti-Müllerian hormone whose concentration when exceeds 3.0 ng/ml, there is evidence that 79% of these patients can be correctly identified as PCOS. The Prostate-Specific Antigen (PSA), a marker of singular importance in patients with prostate cancer, with ultra-sensitive techniques, has been detected in more than 50% of women. In a group of patients with PCOS, circulating levels of PSA are significantly higher than in women without PCOS. The Kiss-1 isolated from the placenta and demonstrated in other tissues, has increased levels that correlate with LH, TT, TL and insulin resistance (IR) in adolescents with PCOS respect to adolescents without PCOS, suggesting that Kiss-1 could be involved in the development of the PCOS in these patients. In some patients with PCOS, they are associated with relevant pathologies, of which the increase in BMI, higher degree of dyslipidemia, central adiposity, IR and Metabolic Syndrome (MeS) have been reported. Those that show a classic phenotype (hyperandrogenism, alteration of the menstrual cycle and polycystic ovaries) these characteristics are of greater frequency and severity than in the other phenotypes, particularly those without hyperandrogenism. Other determinations such as TNFα, interleukins, glucose tolerance test, ApoB, small particles of LDL and Plasminogen Activator Inhibitor-1 have been reported that could be useful to have greater sensitivity in the definition of concomitant pathology in the PCOS. Currently, other markers such as Fetuin-A, Chemerin, Nesfatin-1 Neopterin and Endocannabinoids have been evaluated. The preliminary results suggest to be an important contribution to define MeS and IR in patient with PCOS and to try to determine its prevalence in the different phenotypes of this pathology.
Asunto(s)
Humanos , Femenino , Síndrome del Ovario Poliquístico/complicaciones , Síndrome del Ovario Poliquístico/fisiopatología , Biomarcadores/análisis , Síndrome del Ovario Poliquístico/sangre , Síndrome Metabólico/complicaciones , Dislipidemias/complicaciones , Andrógenos/análisisRESUMEN
Esta revisión fue realizada con el fin de evaluar nuestros resultados de laboratorio así como aquellos de la literatura que constituyen, a nuestro entender, aportes significativos en el síndrome de ovarios poliquísticos (SOP). Nuestro especial énfasis será presentar las limitaciones de las metodologías empleadas por nuestro grupo, comparativamente a las reportadas por otros investigadores. La determinación de andrógenos, en particular de Testosterona (TT), es quizá la de mayor complejidad dado que los resultados con los diferentes inmunoensayos empleados en nuestro medio producen resultados muy variables por los diferentes métodos y aún entre laboratorios que usan la misma metodología. La técnica de referencia es la cromatografía líquida en tándem con espectrometría de masa (LC-MSMS), de difícil aplicación en laboratorios de análisis clínicos debido a su alto costo y la imposibilidad de resolver numerosas muestras. En estudios previos demostramos que de los métodos habitualmente usados para evaluar la TT circulante, solo en 2 inmunoensayos los resultados obtenidos fueron satisfactoriamente validados indirectamente según el criterio del Consenso de los Centros para el Control y Prevención de Enfermedades (CDC, USA) contra LC-MSMS, los cuales fueron comparables a dicha metodología con niveles superiores a 0,5 ng/ml. El SOP puede presentar factores de riesgo aumentados para la enfermedad cardiovascular y la diabetes II. Estos factores no están debidamente categorizados en función de los distintos fenotipos del SOP. Se evaluarán los principales analitos empleados con este objetivo y los nuevos que aporten elementos de mayor especificidad en este sentido
This review was performed in order to evaluate our laboratory results as well as those of the literature that constitute, in our opinion, significant contributions in these pathophysiologies. Our special emphasis will be on presenting the limitations of the methodologies used by our group, compared to those reported by other researchers. The determination of androgens, in particular Testosterone (TT), is perhaps the most complex since the results with the different immunoassays used in our environment produce very variable results by the different methods and even between laboratories that use the same methodology. The reference technique is LC-MSMS, difficult to apply in clinical analysis laboratories because of its high cost and the inability to solve numerous samples. In previous studies, we demonstrated that, in comparison to LC-MSMS with the usual methods for evaluating circulating TT, the results obtained in only 2 immunoassays were satisfactorily validated indirectly according to the criteria of CDC against LC-MSMS, which were comparable to that methodology with levels higher than 0.5 ng/ml. PCOS may have increased risk factors for cardiovascular disease and diabetes II. These factors are not properly categorized according to the different phenotypes of PCOS. The main analytes used for this purpose will be evaluated and new ones that contribute elements of greater specificity in this sense
Asunto(s)
Humanos , Femenino , Síndrome del Ovario Poliquístico/etiología , Síndrome del Ovario Poliquístico/fisiopatología , Testosterona/análisis , Fenotipo , Espectrometría de Masas/métodos , Inmunoensayo/métodos , Cromatografía Liquida/métodosRESUMEN
Upon transition from growth medium to acetate sporulation medium buffered at pH 6.1 with 0.2 M PIPES, Hansenula saturnus showed a respiratory activity which was 88% antimycin A sensitive (1st) and 12% high azide sensitive (3rd), as in acetate complete growth medium. After â 10 h, 3rd respiration declined and oxygen consumption was inhibited by the simultaneous addition of antimycin A and hydroxamate, a situation which lasted until the appearance of the first asci. Later on, 1st and 3rd respiration reappeared and asci formation was completed under these respiratory conditions. The growth in the presence of antimycin A or erythromycin affected only quantitatively the ascospore production and this is because in sporulation medium there was a de novo synthesis of the mitochondrial components of the respiratory chain. Cells which were avoid of 1st respiration but possessed 2nd or 3rd respiration could sporulate, indicating that these alternative respirations also have a role in the process. This was confirmed by the inhibition of sporulation as occurred in the presence of inhibitors of 1st, 2nd and 3rd respiration in sporulation medium.
RESUMEN
In this paper evidence is presented for the mitochondrial localization of the antimycin A (AA) + salicylhydroxamate (SHAM)-insensitive respiration of the yeasts Kluyveromyces lactis, Endomycopsis capsularis and Hansenula saturnus. Such a respiration, which can be sustained by NADH and NADPH but not by succinate, is inhibited by high concentrations of azide. AA + SHAM-insensitive respiration is not phosphorylating and its postulated physiological role is to oxidize NADH.
Asunto(s)
Antifúngicos/farmacología , Antimicina A/farmacología , Consumo de Oxígeno/efectos de los fármacos , Salicilamidas/farmacología , Levaduras/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NAD/metabolismo , NADP/metabolismo , Pichia/efectos de los fármacos , Pichia/metabolismo , Saccharomycetales/efectos de los fármacos , Saccharomycetales/metabolismo , Succinatos/metabolismo , Levaduras/efectos de los fármacosRESUMEN
Increasing the glucose concentration from 0.1 to 10% in exponentially growing cultures of Kluyveromyces lactis CBS 2359 does not repress the antimycin-sensitive respiration (QO2 of 80 microliter O2 . h-1 . mg-1 dry weight) but raises the antimycin-insensitive respiration from 3 to 12 microliter O2 . h-2 . mg-1 dry weight. Antimycin A inhibits the growth of K. lactis on a variety of substrates with the exception of glucose at concentrations equal to or higher than 1% where substantial antimycin-insensitive respiratory rates are induced. It can be concluded that a minimal antimycin-insensitive QO2 is necessary for cellular growth when the normal respiratory pathway is not functional. The antimycin-insensitive respiration elicited by growth in high glucose concentrations is poorly inhibited by hydroxamate and is inhibited by 50% by 90 microM azide or 1 mM cyanide. These concentrations are much higher than those necessary to inhibit cytochrome c oxidase which is not involved in the antimycin-insensitive respiration as was demonstrated by spectral measurements. A pigment absorbing at 555 nm is specifically reduced after addition of glucose to antimycin-inhibited cells. The same pigment is reoxidized by further addition of high concentrations of sodium azide indicating its participation in the antimycin-insensitive, azide-sensitive respiration.
Asunto(s)
Antimicina A/farmacología , Ascomicetos/metabolismo , Glucosa/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Saccharomycetales/metabolismo , Azidas/farmacología , Metabolismo de los Hidratos de Carbono , Proteínas Fúngicas/biosíntesis , Saccharomycetales/crecimiento & desarrollo , Azida Sódica , Cianuro de Sodio/farmacologíaRESUMEN
The KlAAC gene, encoding the ADP/ATP carrier in Kluveromyces lactis, has previously been cloned by complementation of the op1(aac2) mutation of Saccharomyces cerevisiae. We examined the effect of a null mutation of this gene on the phenotype of K. lactis. The consequence of this mutation was found to be multiple. The mutant was respiratory deficient, had an undetectable level of cytochrome a-a3 and b and did not grow on glycerol. The mitochondrial D-lactate ferricytochrome c oxidoreductase activity, as well as the lactate-induced transcription of its gene, KlDLD, was severely reduced. Furthermore, the mutant was unable to grow on galactose, maltose and raffinose. Transcript analysis showed that KlAAC was the only ADP/ATP carrier gene present in K. lactis. The Klaac mutation was fully complemented not only by AAC2, the major gene for the ADP/ATP carrier in S. cerevisiae, but also by AAC1, a gene which is poorly expressed in S. cerevisiae. AAC1 introduced in K. lactis was transcribed to a high level consistent with normal growth on glycerol being restored in the transformed mutant. KlAAC was not subject to control by KlHap2, in contrast to AAC2 which is regulated by the Hap2 complex in S. cerevisiae.
Asunto(s)
Factor de Unión a CCAAT , Eliminación de Gen , Genes Fúngicos , Kluyveromyces/genética , Lactato Deshidrogenasas , Translocasas Mitocondriales de ADP y ATP/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Citocromos/análisis , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Dosificación de Gen , Prueba de Complementación Genética , Glicerol/metabolismo , Kluyveromyces/química , Kluyveromyces/enzimología , Kluyveromyces/metabolismo , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Translocasas Mitocondriales de ADP y ATP/fisiología , Mutagénesis Insercional , Fenotipo , ARN Mensajero/análisis , ARN Mensajero/genética , Saccharomyces cerevisiae/enzimología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
The respiratory activities and the cytochrome spectra from four species belonging to the genus Hansenula have been analysed. The results obtained and described in this paper show that H. glucozyma possesses only the primary, antimycin A-sensitive respiration, H. anomala and H. californica possess primary and secondary (salicylhydroxamate-sensitive) respirations, whereas H. saturnus possesses three respiratory activities (AA-sensitive, SHAM-sensitive, and AA + SHAM-insensitive). The respiratory activity of H. glucozyma is glucose-repressible, whereas the activities of the other species are not. In addition, antimycin A (AA) and erythromycin (ERY) in the culture media differently inhibit the growth of the four species and regulate the respiratory pathways in the species analysed.
Asunto(s)
Pichia/metabolismo , Saccharomycetales/metabolismo , Antimicina A/farmacología , Citocromos/metabolismo , Eritromicina/farmacología , Glucosa/metabolismo , Consumo de Oxígeno , Pichia/efectos de los fármacos , Pichia/crecimiento & desarrollo , Especificidad de la EspecieRESUMEN
A mutation (op1) in the Saccharomyces cerevisiae AAC2 gene, which codes for the most abundant ADP/ATP carrier isoform, results in lack of mitochondrial-dependent growth and in an as yet unexplained petite-negative phenotype. A gene from the petite-negative yeast Kluyveromyces lactis has been isolated by complementing in multicopy the op1 mutation of S. cerevisiae. This gene, designated KIAAC, can complement the petite-negative phenotype of op1 as well as its inability to grow on nonfermentable carbon sources. KIAAC contains a 915-base pair open reading frame coding for a protein of 305 amino acids which shows a high degree of identity to AAC2. The K. lactis ADP/ATP carrier also shares identity with other known ADP/ATP carrier sequences. In particular, the degree of identity of KIAAC is higher with the Neurospora crassa carrier (80.1%) than with AAC1 (76.6%). The nucleotide sequence upstream of the KIAAC coding region was found to contain a long DNA segment with no coding potential, but presenting features of highly regulated promoter sequences.
Asunto(s)
Genes Fúngicos , Kluyveromyces/genética , Translocasas Mitocondriales de ADP y ATP/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Kluyveromyces/enzimología , Datos de Secuencia Molecular , Mutación , Fosforilación Oxidativa , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
Hansenula saturnus is a petite-negative yeast species which displays a different pattern of respiration depending on the age of the cultures. The respiration is sensitive to antimycin A (AA) in the early exponential phase, is sensitive to the simultaneous addition of AA and salicylhydroxamic acid (SHAM) in the middle exponential phase and is sensitive to SHAM in the late exponential and stationary phase. The three respiratory activities are all associated to the mitochondrial fraction. The presence of AA in the growth medium determines the induction of the AA + SHAM-insensitive respiration which is 50% inhibited by 5 mM azide. On the contrary, the presence of erythromycin in the growth medium, which inhibits mitochondrial protein synthesis in this yeast species and the synthesis of cytochromes aa3 and b, totally prevents the appearance of AA + SHAM-insensitive respiration. Moreover, the antibiotic affects cell viability, suggesting a role of the mitochondrial protein synthesis in the cell cycle of H. saturnus.
Asunto(s)
Ascomicetos/metabolismo , Pichia/metabolismo , Antimicina A/farmacología , Eritromicina/farmacología , Mitocondrias/efectos de los fármacos , Pichia/efectos de los fármacos , Pichia/crecimiento & desarrollo , Salicilamidas/farmacologíaRESUMEN
Introducción: El objetivo de este estudio fue evaluar Los niveles de Testosterona (T), T libre TL, DHEAs y Androstanodiol glucuronidato (A2G) mujeres hirsutas con ciclos menstruales (CM) regulares en la fase folicular (FF) y en una muestra tomada entre -5 a -10 días premenstrual (FL) a los efectos de 1) poder definir bioquímicamente el tipo de hirsutismo y 2) determinar si el aumento de Progesterona modifica los niveles de los andrógenos. Materiales y Métodos: En 65 mujeres hirsutas con CM regulares se determinó en FF los niveles de T, A2G, y DHEAs por RIE y TL calculada por la ecuación de la ley de acción de masas, y en la FL los niveles de P4. En 28 de las 65 pacientes, en la FL se repitió el perfil androgénico Resultados: Los niveles de T correlacionaron, en todos los casos, con los de TL. En 51 de las pacientes los niveles de P4 fueron ovulatorios, 25 de las cuales tuvieron normales los andrógenos evaluados (Hirsutismo Idiopático) De las 26 pacientes restantes, en 2 tenían T aumentada, en 4 la DHEAS. Se obtuvieron 2 parámetros aumentados en los siguientes casos; en 2 la DHEAs y el A2G, en 1 la T y la DHEAs y en1 la T y el A2G. En 4 pacientes se obtuvieron incremento de los 3 parámetros. Estas pacientes corresponden a Hiperandrogénicas ovulatorias. Las 12 restantes de estas 26 hirsutas tenían solamente el A2G aumentados. Dado que éste constituye la expresión periférica de la 5alfa reductasa, las mismas podrían incluirse en el grupo de hiperandrogénicas ovulatorias por aumento local de DHT. En 14 de las 65 pacientes los niveles de P4 fueron compatibles con ciclos anovulatorios correspondiendo a pacientes con Síndrome de Ovario Poliquístico (SOP). En 6 de ellas se constató aumento de 1, 2 o los 3 parámetros evaluados (SOP hiperandrogénicos), en las restantes 6 pacientes los niveles androgénicos fueron normales (SOP con hirsutismo clínico). El A2G aumentó significativamente en FL en las mujeres con ciclos ovulatorios (4.89±2.19 vs 3.36±2.38 ng/ml en FL y FF, respectivamente). En las anovulatorias las diferencias no fueron significativas (4.32±3.16 vs 4.69±4.54 ng/ml en FL y FF, respectivamente. Estos resultados indican que la P4 podría inducir un incremento del A2G. Dado que la T no se modificó en la FL respecto a FF (0.28±0.22 vs 0.30±0.25ng/ml en hirsutas ovulatorias y 0.47±0.32 vs 0.42±0.23 en hirsutas anovulatorias) es posible que la P4 aumente el A2G por un camino distinto a la de la T y DHT Conclusiones: En base a estos resultados podemos concluir que la determinación de A2G podría ser empleada como parámetro complementario en el estudio del hiperandrogenismo debiendo realizarse en FF dado que en FL podría ser el resultado del metabolismo de hormonas no androgénicas.
Introduction: The aim of the present study was to evaluate the circulating levels of Testosterone (T), free T (TL), DHEAs and Androstanediol glucuronide (A2G) in hirsute women with regular menstrual cycles (CM) in follicular phase (FF), and in a samples obtained 5 to 10 days before the next menstrual bleeding (FL), in order to 1) biochemically define type of hirsutism and 2) determine whether the increase in progesterone (P4) induces changes in androgen levels. Materials and Methods: Sixty five hirsute women with regular CM were studied. FF levels of T, A2G and DHEAs were determined by RIA, and TL by mass law calculation. FL levels of P4 were measured by RIA. In 28 of the 65 patients the androgen profile was also evaluated in FL. Results: The levels of T correlated in every case with those of TL. In 51 patients P4 levels were ovulatory. Twenty five of them showed normal androgen levels (Idiopathic hirsutism). From the remaining 26 patients, 2 had increased T, and 4 had increased DHEAs. Two parameters were found increased in the following cases: DHEAs and A2G in 2, T and DHEAs in 1, and T and A2G in 1. All the 3 parameters were found increased in 4 cases. These patients were ovulatory hiperandrogenic women. The remaining 12 of these 26 hirsute women had only A2G increased. Since this steroid is the peripheral expression of the 5alpha reductase activity, these women could be included in the ovulatory hiperandrogenic group because of a local increase in DHT. In 14 of the 65 patients the levels of P4 correlated with anovulatory cycles corresponding to Polycystic Ovarian Syndrome (SOP). In 6 of them an increase of 1, 2 or the 3 parameters were observed (Hiperandrogenic SOP); in the remaining 6 patients androgen levels were normal (SOP with clinical hirsutism). FL A2G significantly increased in women with ovulatory cycles (4.89±2.19 vs 3.36±2.38 ng/ml in FL and FF, respectively. Differences were no significant in the anovulatory patients (4.32±3.16 vs 4.69±4.54 ng/ml in FL and FF, respectively. These results indicate that P4 could induce an increase in A2G. Since T did not change in FL respect to FF (0.28±0.22 vs 0.30±0.25ng/ml in ovulatory hirsute and 0.47±0.32 vs 0.42±0.23 in anovulatory hirsute) it is possible that P4 increases A2G through a pathway different than that of T and DHT. Conclusions: Based on these results we conclude that A2G could be used as a complementary parameter in the study of hiperandrogenism, only in FF since in FL, it could be the result of the metabolism of non-androgenic hormones.