Alphaviral equine encephalomyelitis (Eastern, Western and Venezuelan).

Summary Alphaviral equine encephalomyelitis is a mosquito-borne infection that causes severe neurological disease and fatalities in horses and humans in the Americas. Consequently, the equine alphaviruses (Eastern, Western and Venezuelan) are of considerable concern worldwide and are notifiable to the World Organisation for Animal Health. In addition, these diseases are considered a potent potential biological weapon, emphasising the need to develop an effective vaccine. Alphaviral equine encephalomyelitis is caused by Eastern equine encephalomyelitis virus (EEEV), Western equine encephalomyelitis virus (WEEV) or Venezuelan equine encephalomyelitis virus (VEEV), which are related members of the Alphavirus genus in the Togaviridae family. Although related, the three viruses are genetically and antigenically distinct. The disease is characterised by fever, anorexia, depression and clinical signs of encephalomyelitis, and may be fatal in up to 90% of cases, for both humans and horses, particularly in the case of EEE. Surviving horses develop lifelong immunity but may have permanent neuropathology. The aim of this paper is to analyse the scientific information available on the evolution of EEE, WEE and VEE, and any potential vaccines.

Control of canine rabies in developing countries: key features and animal welfare implications.

Over 90% of human deaths from rabies worldwide are caused by dog bites. Mass vaccination, along with the effective control of dog populations, has been used successfully in industrialised countries to control this disease. A lower success rate in developing countries is due to a number of factors, including vaccination campaigns that do not cover a sufficient number of animals or reach all communities, and a wide biodiversity that increases the number of reservoirs of the rabies virus. Educational programmes are needed, which focus on the commitment involved when acquiring a domestic animal, stating clearly what is required to provide it with a good quality of life. New technologies developed in the industrialised world will not always be successful in less developed countries. Approaches must be adapted to the particular conditions in each country, taking cultural and socio-economic issues into account. Authorities must promote research on dog population dynamics, the development of non-invasive methods to control dog populations and the most efficient, stable and low-cost options for vaccination. Under the One Health model, it is hoped that dog-transmitted human rabies will be accorded high priority as a zoonosis by human health authorities, international authorities and donor agencies to support ambitious eradication goals, particularly those being set in South-East Asia. Well-designed and adequately resourced vaccination programmes, based on the World Organisation for Animal Health (OIE) guidelines, will have significant animal welfare benefits, due to the availability of improved vaccines (in terms of efficacy, duration of immunity, ease of administration and lower cost), advances in dog population management and the more widespread implementation of the OIE Guidelines on Stray Dog Control. Animal welfare benefits include not only the elimination of pain and suffering caused by the clinical disease itself, but also the avoidance of the indirect impact of inhumane culling when methods are used that have not been approved by the OIE.

Experimental infection of Artibeus intermedius bats with serotype-2 dengue virus.

Dengue fever is caused by a flavivirus that primarily infects humans and Aedes sp. mosquitoes. However, viral replication in wild animals other than non-human primates has been scarcely studied. In this report, the susceptibility of Artibeus intermedius frugivorous bat to serotype-2 dengue virus (DENV-2) infection was tested. Twenty-three bats were intraperitoneally inoculated with different viral loads of DENV-2 (New Guinea-C strain). Forty-three percent of the infected bats developed bruises on the chest or on the wings. Histological analyses showed structural alterations in the spleen and bleeding in liver and intestine, but the virus was not detected by RT-PCR in any of the analyzed tissues, and it was found in only one bat (kidney) by semi-nested RT-PCR. In sera, the viral RNA was detected by semi-nested RT-PCR in 39% of bats, but only 8% of bats seroconverted. Overall, these data indicate that DENV-2 replicates poorly in these bats, suggesting they are not suitable hosts to this virus.

Evaluation of four Agrobacterium tumefaciens strains for the genetic transformation of tomato (Solanum lycopersicum L.) cultivar Micro-Tom.

KEY MESSAGE : Agrobacterium tumefaciens strains differ not only in their ability to transform tomato Micro-Tom, but also in the number of transgene copies that the strains integrate in the genome. The transformation efficiency of tomato (Solanum lycopersicum L.) cv. Micro-Tom with Agrobacterium tumefaciens strains AGL1, EHA105, GV3101, and MP90, harboring the plasmid pBI121 was compared. The presence of the nptII and/or uidA transgenes in regenerated T(0) plants was determined by PCR, Southern blotting, and/or GUS histochemical analyses. In addition, a rapid and reliable duplex, qPCR TaqMan assay was standardized to estimate transgene copy number. The highest transformation rate (65 %) was obtained with the Agrobacterium strain GV3101, followed by EHA105 (40 %), AGL1 (35 %), and MP90 (15 %). The mortality rate of cotyledons due to Agrobacterium overgrowth was the lowest with the strain GV3101. The Agrobacterium strain EHA105 was more efficient than GV3101 in the transfer of single T-DNA insertions of nptII and uidA transgenes into the tomato genome. Even though Agrobacterium strain MP90 had the lowest transformation rate of 15 %, the qPCR analysis showed that the strain MP90 was the most efficient in the transfer of single transgene insertions, and none of the transgenic plants produced with this strain had more than two insertion events in their genome. The combination of higher transformation efficiency and fewer transgene insertions in plants transformed using EHA105 makes this Agrobacterium strain optimal for functional genomics and biotechnological applications in tomato.

New rabies virus variant found during an epizootic in white-nosed coatis from the Yucatan Peninsula.

In February 2008, three white-nosed coatis (Nasua narica) were found dead in a recreational park in Cancun, Mexico. The diagnosis of rabies virus (RABV) infection was confirmed by direct immunofluorescence test. The phylogenetic analysis performed with the complete RABV nucleoprotein gene positioned this isolate close to a sequence of a human rabies case reported during 2008 from Oaxaca, Mexico, sharing 93% similarity. In turn, these two variants are related to another variant found in rabid Tadarida brasiliensis mexicana bats across North America. Anti-RABV neutralizing activity (1.3 IU/ml) was found in the serum of one white-nosed coati captured with another five that cohabited with the dead animals. Enhanced rabies surveillance and pathogenesis studies should be conducted in coatis and insectivorous bats of the region to clarify the role of these species as potential emergent or long-term unidentified RABV reservoirs.

Bioequivalencia entre dos formulaciones de claritromicina comprimidos de liberación modificada existentes en el mercado chileno / Bioequivalence of two brands of clarithromycin in modified-release tablets available in the chilean pharmacopeia

Se realizó un estudio comparativo de biodisponibilidad, para evaluar la bioequivalencia entre dos formulaciones de claritromicina de 500 mg en comprimidos de liberación modificada, una nacional (Pre-Clar UD (R)), y el innovador del mercado (Klaricid UD (R)). Se utilizó un ensayo microbiológico para determinar las concentraciones plasmáticas del antimicrobiano. El ensayo está basado en la correlación entre la inhibición del crecimiento bacteriano y la concentración plasmática de claritromicina. Un total de 16 voluntarios, jóvenes sanos, no fumadores, participaron y completaron el protocolo del estudio, el cual fue aprobado por el Comité de Ética de la Facultad de Medicina de la Universidad de Chile. Los parámetros farmacocinéticos de: concentración plasmática máxima (Cmáx,), tiempo de vida media (t1/2), área bajo la curva de concentraciones plasmáticas versus tiempo desde cero a infinito (ABC0-Ñ) ,constante de velocidad de absorción (Kabs), no mostraron diferencias estadísticamente significativas entre los productos utilizados. De acuerdo a los criterios recomendados por la FDA y en base a nuestros resultados, se concluye que las formulaciones Pre-Clar UD® y Klaricid UD® son bioequivalentes, asumiéndose que tendrán igual eficacia clínica.

Bioequivalencia entre dos formulaciones de claritromicina en suspensión pediátrica existentes en el mercado chileno / Bioequivalence evaluation of two brands of clarithromycin suspension available in the chilean pharmacopeia

Se realizó un estudio comparativo de biodisponibilidad, para evaluar la bioequivalencia entre dos formulaciones de claritromicina en suspensión de 250 mg/5 mL, una nacional (Pre-Clar(R)), y el innovador del mercado (Klaricid(R)). En ambos casos, se administró una dosis de 500 mg. Se utilizó un ensayo microbiológico para determinar las concentraciones plasmáticas del antimicrobiano. El ensayo está basado en la correlación entre la inhibición del crecimiento bacteriano y la concentración plasmática de claritromicina. Un total de 12 voluntarios, jóvenes sanos, participaron y completaron el protocolo del estudio, el cual fue aprobado por el Comité de Ética de la Facultad de Medicina de la Universidad de Chile. Los parámetros farmacocinéticos de concentración plasmática máxima (Cmáx), tiempo de vida media (t1/2), área bajo la curva concentraciones plasmáticas versus tiempo, desde cero a infinito (ABC0-Ñ) y la constante de velocidad de absorción (Kabs), no mostraron diferencias estadísticamente significativas entre los productos utilizados. De acuerdo a los criterios recomendados por la FDA y sobre la base de nuestros resultados, se concluye que las formulaciones en suspensión pediátrica de 250 mg/5 mL Pre-Clar® y Klaricid® son bioequivalentes asumiéndose que tendrán igual eficacia clínica.

Seasonal changes in the activity of cytochrome P450(C17) from the testis of Bufo arenarum.

In Bufo arenarum, the biosynthesis of testosterone and 5alpha-dihydrotestosterone takes place through a complete 5-ene pathway, 5-androsten-3beta,17beta-diol being the immediate precursor of testosterone. Besides androgens, testes are able to synthesise 5alpha-pregnan-3,20-dione and several 3alpha and 20alpha reduced derivatives. During the breeding season, steroid biosynthesis turns from androgen to C21-steroid production. As a consequence, the cytochrome P450 17-hydroxylase, C17,20-lyase (CypP450(c17)) could be a key enzyme in that metabolic shift. The present study demonstrates that in testes of B. arenarum, CypP450(c17) co-localises with glucose-6-phosphatase in the microsomal fraction. CypP450(c17) possesses more affinity for pregnenolone than for progesterone in both non-reproductive (Km = 43.76 +/- 4.63 nM and 2,170 +/- 630 nM, respectively) and reproductive (Km = 37.46 +/- 4.19 nM and 3,060 +/- 190 nM, respectively) seasons. These results could explain the predominance of the 5-ene pathway for testosterone biosynthesis. Toad CypP450(c17) activity is higher in the non-reproductive period than the reproductive period, suggesting that this enzyme is an important factor in toad steroidogenic changes. Animals in reproductive conditions showed a significant reduction in circulating androgens. This is in agreement with the decrease in Vmax of cytochrome P450 17-hydroxylase activity, enhancing the physiological relevance of these in vitro results.

In vitro hCG and human recombinant FSH actions on testicular steroidogenesis in the toad Bufo arenarum.

In order to study the regulation of testicular steroidogenesis in the toad Bufo arenarum, the effect of gonadotropins (hCG and hrFSH) on steroidogenic enzymes was determined using an in vitro system. 3beta-Hydroxysteroid dehydrogenase/isomerase activity was not affected by any of the gonadotropins, at any of the concentrations used. In contrast, 5alpha-reductase activity was strongly reduced by both hCG and hrFSH. Human chorionic gonadotropin inhibited the activity of cytochrome P450 17alpha-hydroxylase-C(17-20) lyase (P450(c17)), only at the highest concentration used, while hrFSH strongly reduced P450(c17) activity at all the doses assayed. In conclusion, these data suggest that LH (hCG) and FSH regulate steroidogenic enzymes such as 5alphaRed and P450(c17). The results also suggest that FSH could be involved in the regulation of the change in steroidogenesis undergone by the testis during the breeding season. In turn, the inhibition of P450(c17) activity could result in a reduction of androgen production and an increment of C21 steroids.

Seasonal changes in testicular steroidogenesis in the toad Bufo arenarum H.

The biosynthesis of androgens in Bufo arenarum takes place through the 5-ene pathway that includes 5-androstane-3beta,17beta-diol as intermediate in testosterone biosynthesis. Besides testosterone and 5alpha-dihydrotestosterone, testes are able to synthesize 5alpha-pregnan-3,20-dione and several 3alpha- and 20alpha-reduced derivatives. Steroid biosynthesis changes during the breeding period (spring and early summer), turning from androgen to C21 steroid production. During the reproductive season, the production of progesterone, 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one, and 5alpha-pregnan-3,20-dione increases significantly. The function of most of these steroids in amphibians remains unknown. However, 5alpha-androstan-3alpha,17beta-diol and 3alpha-hydroxy-5alpha-pregnan-20-one were shown to be neuroactive in mammals, modulating sexual behavior. Thus, 5alpha/3alpha-reduced steroids could be involved in the regulation of the reproductive behavior in B. arenarum, a species with a dissociated reproductive pattern. Percentage contribution of each enzymes to the total metabolism reveals that neither 3beta-hydroxysteroid dehydrogenase/isomerase nor 5alpha-reductase change throughout the reproductive cycle. However, a strong reduction in 17-hydroxylase-C(17-20) lyase activity occurs in the reproductive season, suggesting that this enzyme could represent a key enzyme in the regulation of the seasonal change of steroidogenesis. Also, 3alpha-hydroxysteroid dehydrogenase and 20-hydroxysteroid dehydrogenase activities increase during the reproductive period, implying that steroid metabolism is clearly focused on C21-reduced steroids.

Effects of different steroid-biosynthesis inhibitors on the testicular steroidogenesis of the toad Bufo arenarum.

Testis fragments from Bufo arenarum were incubated with [7(n)-(3)H]pregnenolone (P5), [1,2-(3)H]dehydroepiandrosterone (DHEA) and [1,2,6.7-(3)H]testosterone (T), and different steroid-biosynthesis inhibitors. The inhibitors used were: cyanoketone (CNK), spironolactone (SPNL) and finasteride (FIN). CNK significantly increased the recovery of 3beta-hydroxy-5-ene steroids while SPNL reduced the metabolism of P5 and the production of C19-steroids. The metabolism of C19-substrates was only modified by CNK, which reduced the transformation of DHEA without modifying the metabolism of T. To determine the degree of inhibition exerted by the inhibitors used, the activities of the enzymes were estimated as the percentage of their contribution to the total steroid metabolism. CNK strongly inhibited the activity of hydroxysteroid dehydrogenase/isomerase if its contribution was estimated using both P5 and DHEA. If the analysis was made considering both activities associated to cytochrome P450 17chi-hydroxylase, C17-20 lyase (P450c17), it became evident that SPNL inhibited both of them. The percent contribution of 17beta-hydroxysteroid dehydrogenase (17betaHSD) activity diminished in the presence of CNK only if it was estimated considering P5 and DHEA metabolism. SPNL produced a significant inhibition of 17betaHSD when its contribution was estimated considering P5 metabolism. However, SPNL was insufficient if DHEA or T were considered. The effect of SPNL on the contribution of 17betaHSD could be due to the reduction of C19-substrates. The activity of 5chi-reductase was inhibited by CNK only if results from P5 and DHEA were considered.

[Long and medium-term neuropsychological sequelae of bone marrow transplantation in pediatric patients with hematological disease]. / Secuelas neuropsicológicas a medio y largo plazo del trasplante de medula ósea en pacientes con enfermedades hematológicas.

BACKGROUND: Few studies exist on the neuropsychological sequelae of bone marrow transplantation (BMT) in pediatric patients. Published results show considerable discrepancies although studies of children with acute lymphoblastic leukemia undergoing high doses of cranial radiotherapy report short- and long-term loss of cognitive ability. OBJECTIVES: To determine the effects of BMT and the effect of anxiety on the pre-BMT assessment in a group of children with severe hematological disease treated in our center. METHODS: We performed a descriptive, prospective, longitudinal study of 54 children, aged 4-15 years, who were treated between 1987 and 1995. Twenty-two children were evaluated before and after BMT by means of the Weschler Intelligence Scale. To control for the effect of anxiety on the pre-BMT scores, the patients were divided into two groups according to the scores obtained in this test (group 1: IQ score 100; group 1: IQ100). RESULTS: Comparison of pre- and post-BMT scores for both groups revealed no significant differences. However, comparison of the results between groups revealed that group I scored lower in the post-BMT test than in the pre-BMT test while group I scored higher in the post-BMT test than in the pre-BMT test. CONCLUSIONS: Although comparison between the pre- and post-BMT results obtained from the whole sample showed no differences that indicated post-treatment sequelae, treatment-induced anxiety may have influenced the pre-BMT score.

Secuelas neuropsicológicas a medio y largo plazo del trasplante de medula ósea en pacientes con enfermedades hematológicas / Long and medium-term neuropsychological sequelae of bone marrow transplantation in pediatric patients with hematological disease

Antecedentes: Existen pocos estudios sobre las secuelas neurocognitivas del trasplante de medula ósea (TMO) en pacientes pediátricos, y sus resultados a menudo son divergentes, aunque los estudios con niños enfermos de leucemia sometidos a altas dosis de radioterapia craneal muestran pérdida de capacidad cognitiva a medio y largo plazo. Objetivos: Conocer los efectos del TMO en un grupo de niños con graves enfermedades hematológicas tratados en nuestro centro, y el impacto de la ansiedad en la evaluación previa al TMO. Métodos: Se realizó un estudio descriptivo prospectivo y longitudinal, desde 1987 hasta 1995 con un grupo 54 niños, de entre 4 y 15 años. De este grupo 22 niños se evaluaron antes y después del TMO mediante la Escala de Inteligencia de Wechsler para niños (Wechsler Intelligence Scale, WIS). Con el fin de controlar el impacto de la ansiedad sobre el rendimiento de la evaluación previa al TMO se dividieron los pacientes en 2 grupos según la puntuación obtenida en dicha evaluación: grupo 1, cociente de inteligencia total (CIT) menor o igual a 100; grupo 2, CIT mayor de 100. Resultados: En la comparación entre los resultados de los 2 grupos pretrasplante y postrasplante del grupo inicial no se observaron diferencias significativas, pero al comparar los resultados por grupos, se observó un descenso significativo en el grupo 2, el que había puntuado más alto en la evaluación previa al TMO, mientras que el grupo 1 aumentaba su rendimiento. Conclusiones: Si bien en una primera comparación entre los resultados pretrasplante y postrasplante de la muestra no parece existir diferencias que muestren secuelas postratamiento, la ansiedad producida por el mismo pudo haber influido en la evaluación pretrasplante, de tal forma que enmascarase los efectos de éste (AU)

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H.

3beta-hydroxysteroid dehydrogenase 5-ene isomerase (3betaHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3betaHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K(m)=0.17+/-0. 03 and 1.02 microM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V(max) while K(m) was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K(i) was calculated from replot of Dixon's slope for each substrate concentration. K(i) from the intercept and the slope of this replot were similar (0.276+/-0.01 and 0.263+/-0.02 microM) and higher than the K(m) for DHEA. The K(m) and K(i) values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V(max) while K(m) values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.

Human chorionic gonadotropin-induced spermiation in Bufo arenarum is not mediated by steroid biosynthesis.

This study employed an in vitro system to identify potential steroidal mediators of spermiation in Bufo arenarum. Testicular fragments were incubated for 2 h at 28 degrees. Spermiation was induced by 10 IU human chorionic gonadotropin (hCG) and the effect of different inhibitors of steroid biosynthesis was analyzed. Cyanoketone (10(-5)-10(-6) M), an inhibitor of 3-oxo-4-ene steroid biosynthesis, did not block hCG-inducing activity even when biosynthesis of 3-oxo-4-ene steroids and its reduced metabolites was inhibited by 95%. Aminogluthetimide at a concentration that inhibited testosterone biosynthesis (10(-4) and 10(-5) M) did not alter hCG actions. Similar results were obtained with spironolactone, an inhibitor of 17alpha-hydroxylase/17-20 lyase activity. No spermiation-inducing activity was found with different steroids (progesterone, 17-hydroxypregnenolone, 17, 20alpha/beta-dihydroxy-4-pregnene-3-one, estradiol, testosterone, etc.). It is concluded that spermiation induced by hCG is not steroid mediated in B. arenarum.

Uptake and incorporation of docosahexaenoic acid (DHA) into neuronal cell body and neurite/nerve growth cone lipids: evidence of compartmental DHA metabolism in nerve growth factor-differentiated PC12 cells.

Docosahexaenoic acid (DHA) accumulates in nerve endings of the brain during development. It is released from the membrane during ischemia and electroconvulsive shock. DHA optimizes neurologic development, it is neuroprotective, and rat adrenopheochromocytoma (PC12) cells have decreased PLA2 activity when DHA is present. To characterize DHA metabolism in PC12 cells, media were supplemented with [3H]DHA or [3H]glycerol. Fractions of nerve growth cone particles (NGC) and cell bodies were prepared and the metabolism of the radiolabeled substrates was determined by thin-layer chromatography. [3H]glycerol incorporation into phospholipids indicated de novo lipid synthesis. [3H]DHA uptake was more rapid in the cell bodies than in the NGC. [3H]DHA first esterified in neutral lipids and later in phospholipids (phosphatidylethanolamine). [3H]glycerol primarily labeled phosphatidylcholine. DHA uptake was compartmentalized between the cell body and the NGC. With metabolism similar to that seen in vivo, PC12 cells are an appropriate model to study DHA in neurons.

Pregnenolone and progesterone metabolism by the testes of Bufo arenarum.

Sliced testis tissue from Bufo arenarum was incubated in the presence of [3H]pregnenolone. Testis fragments were also used for double isotope experiments using [3H]pregnenolone and [14C]progesterone. Specific activities were equated with the addition of radioinert pregnenolone. When yields of radiometabolites were analysed, pregnenolone was found to be a good precursor for C19 steroids such as dehydroepiandrosterone, 5-androsten-3 beta, 17 beta diol, testosterone, 5 alpha-dihydrotestosterone and a C21 steroid, 5 alpha-pregnan-3,20 dione. Progesterone mainly converts to 5 alpha-pregnan-3,20 dione, a steroid with unknown function in amphibians. The 5-ene pathway, including 5-androsten-3 beta, 17 beta diol as intermediate, could be predominant for androgen biosynthesis. Testes bypass not only progesterone but also androstenedione for testosterone biosynthesis.

[Diagnosis of breast tumors by scintigraphy versus mammography]. / Diagnóstico de tumores mamarios por centelleografía versus mastografía.

OBJECTIVE: To compare the efficacy of the breast scintigraphy with 99mTc-MIBI in the detection of breast lesions versus mammography. DESIGN: Cross sectional study. SETTING: A concentration oncologic hospital in Mexico City. PATIENTS: 56 women with a biopsy performed due to radiologic suspicion of mammary cancer were included as well as 10 normal women. MAIN PROCEDURES: Mammography, breast scintigraphy with 99mTc-MIBI, and biopsy were performed. RESULTS: Sensitivity of breast scintigraphy was 100% specificity 94.6%, PPV 90% and NPV 100%; sensitivity of the mammography was 100%, specificity 54% PPV 52.7%, and NPV 100%. CONCLUSIONS: Breast scintigraphy with 99mTc-MIBI showed a higher sensitivity and specificity than those of mammography. It was highly reliable for the diagnosis of mammary gland malignant lesions.

Subcellular localization of 3 beta hydroxysteroid dehydrogenase isomerase in testis of Bufo arenarum H.

3 Beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones and is usually considered to be mainly microsomal, although there is a dual distribution of the enzyme in toad interrenals. The present study demonstrates that in the testicular tissue, as in interrenals of Bufo arenarum H., 3 beta HSD/I is both mitochondrial and microsomal. The conversion of dehydroepiandrosterone to androstenedione takes place only in microsomes while pregnenolone is converted to progesterone in both microsomes and mitochondria. Kinetic constants of 3 beta HSD/I were determined by the oxidation of pregnenolone and dehydroepiandrosterone. The preferred substrate of the microsomal 3 beta HSD/I enzyme was dehydroepiandrosterone (K(m) = 0.17 microM and 0.53 microM for dehydroepiandrosterone and pregnenolone, respectively) not only during the breeding season but also in the non-breeding period (K(m) = 0.49 microM and 2.9 microM for dehydroepiandrosterone and pregnenolone, respectively).

Mitochondrial localization of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase in interrenals of the toad Bufo arenarum H.

The enzymatic activity of 3 beta-hydroxysteroid dehydrogenase 5-ene isomerase (3 beta HSD/I) catalyzes an essential step in the biosynthesis of steroid hormones including progesterone, mineralocorticoids, glucocorticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal. The present study demonstrates that in the interrenal of Bufo aernarum H., 3 Beta HSD/I activity localizes in mitochondria and micromes. It also shows that the two distinct pathways to aldosterone previously demonstrated for interrenals of B. arenarum H. exhibited differential subcellular localizations, microsomal for the 4-ene route and mitochondrial for the 5-ene route. Kinetic constants of 3 Beta HSD/I were determined for the oxidation of pregnenolone and the recently described 3 Beta-hydroxy analogue of aldosterone (3 Beta AA). The preferred substrate of the mitochondrial 3 Beta HSD/I enzyme was 3 Beta AA (Km = 0.7 microM and 14.0 microM for 3 Beta AA and pregnenolone, respectively). However, the microsomal enzyme has a greater affinity for pregnenolone (Km = 0.8 microM) than for 3 Beta AA (Km = 17.0). Enzymes from both localizations have similar nucleotide (NAD+) requirements, activities being higher in summer. This dual localization opens novel possibilities for the regulation of interrenal functions.