Ferredoxin-dependent glutamate synthase: involvement in ammonium assimilation in Haloferax mediterranei.

Glutamate synthase (GOGAT) is one of the two important enzymes involved in the ammonium assimilation pathway glutamine synthetase (GS)/GOGAT, which enables Hfx. mediterranei to thrive in media with low ammonium concentration or containing just nitrate as single nitrogen source. The gene coding for this enzyme, gltS, has been sequenced, analysed and compared with other GOGATs from different organisms from the three domains of life. According to its amino acid sequence, Hfx. mediterranei GOGAT displays high homology with those from other archaeal halophilic organisms and with the bacterial alpha-like subunit. Hfx. mediterranei GOGAT and GS expression was induced under conditions of ammonium restriction. The GOGAT protein was found to be a monomer with a molecular mass of 163.78 kDa, which is consistent with that estimated by gel filtration, 198 ± 30 kDa. The enzyme is highly ferredoxin dependent: activity was only observed with one of the two different 2Fe-2S ferredoxins chromatographically isolated from Hfx. mediterranei. The enzyme also displayed typical halophilic behaviour, being fully stable, and producing maximal activity, at salt concentrations from 3 to 4 M NaCl, pH 7.5 and a temperature of 50 °C.

Mechanical allodynia in neuropathic pain. Where are the brain representations located? A positron emission tomography (PET) study.

BACKGROUND: Brain areas involved in nociception have been repeatedly investigated. Therefore, brain responses to physiological pain conditions are well identified. The same is not true for allodynic pain in patients with neuropathic pain since the cortical reorganizations that are involved in the conversion of non-noxious stimuli into painful sensations still remain unknown. METHODS: The present positron emission tomography (PET) study enrolled 19 patients with dynamic mechanical allodynia to brushing or to cold rubbing of the skin. PET activations during allodynic stimulation were compared to those obtained with the same innocuous stimulation applied outside the neuropathic pain area (control). In a second comparison, they were compared with responses to a noxious heat stimulation applied outside the neuropathic pain area (experimental pain). RESULTS: Common responses to allodynia and control stimulations were found in contralateral SI, SII and insula and in ipsilateral cerebellum. Not surprisingly, heat pain condition was associated with activations in contralateral prefrontal and SII cortices and, bilaterally, in the anterior insular cortices. Distinctive cortical responses between control and allodynic conditions were restricted to one activation within the contralateral anterior insula, a region also activated by experimental heat pain. CONCLUSIONS: The insular subdivision was inappropriately activated considering the innocuous nature of the stimulus, but adequately activated with regard to pain-evoked sensation. Subcortically, the hypothesis of reorganization at any level of the somatosensory and pain pathways underlying such insular activity was supported by the observed shift of thalamic activation from a lateral-posterior to an anterior-medial position.

Modulations of pain sensations.

Representation of time may affect pain perception. We investigated a group of volunteers looking at different clocks while they were being exposed to the same intensity of pain in two experiments. In one case, they saw the actual time, while in the other, they gazed at a clock that made it seem like the stimulation was shortened, even though it wasn't. These results show that simply believing that time is on your side can make anything more bearable. The results were not influenced by the color of the clock (red or green), or the presence of indexes such as (sad or smiling) smileys. The effects were maximal for high intensities of stimulation (pain threshold +1°C) if the stimulation lasted for at least 25s but were absent if the stimulation was short (15 min). These results suggest that pain modulation by time context is mainly available for long and intense painful stimulations. The right upper and posterior parietal cortex may support this effect. These findings are discussed with regard to previous literature of pain modulations but also with regard to the concept of the "pain matrix", its inputs and the temporal dynamics of its constitutive responses.

Intensa retención de 99mTc-MIBI en la fase tardía como causa de error diagnóstico en la gammagrafía combinada paratiroides-tiroides / High retention of 99mTc-MIBI in delayed phase as a pitfall in the combined parathyroid-thyroid scintigraphy

La gammagrafía de paratiroides con 99mTc-sestaMIBI es muy sensible en el diagnóstico y localización del adenoma de paratiroides. Sin embargo, se pueden producir errores diagnósticos debido a la coexistencia de lesiones tiroideas captantes de MIBI o en pacientes con cirugía tiroidea previa. Para intentar detectar dichas alteraciones tiroideas se realiza una gammagrafía con 99mTc-pertecnetato comparativa el mismo día. Ocasionalmente, como en nuestro caso, la lesión paratiroidea puede presentar una captación inicial y retención tardía de MIBI lo suficientemente importantes como para ser visualizadas en la gammagrafía tiroidea realizada a continuación, dando la falsa apariencia de una lesión captante también de pertecnetato. Este hecho ha sido llamado «shine through». Para evitarlo se pueden realizar las gammagrafías paratiroidea y tiroidea en días distintos. A nosotros nos resultó también de gran utilidad la comparación con ecografía y la PAAF de la lesión con determinación de PTH y tiroglobulina en el líquido aspirado(AU)

Dual phase parathyroid scintigraphy with 99mTc-sestaMIBI is a very sensitive technique in the preoperative localization and diagnosis of parathyroid adenoma. However, pitfalls have been reported in patients with thyroid nodules with MIBI uptake or with previous thyroid surgery. To solve this problem, a thyroid scintigraphy with 99mTc-pertechnetate is usually performed following the parathyroid study. Occasionally, as in our patient the parathyroid lesion may show high MIBI uptake and delayed washout that interfere with the subsequent thyroid scintigraphy giving the false appearance of a pertechnetate avid lesion. This has been called the «shine through» effect. To avoid it, the parathyroid and thyroid scintigraphies can be performed on separate days. We have also found it useful to compare our results with that of ultrasound and fine needle aspiration puncture with measurement of the parathyroid hormone (PTH) and thyroglobulin in the aspirated material(AU)

[High retention of (99m)Tc-MIBI in delayed phase as a pitfall in the combined parathyroid-thyroid scintigraphy]. / Intensa retención de (99m)Tc-MIBI en la fase tardía como causa de error diagnóstico en la gammagrafía combinada paratiroides-tiroides.

Dual phase parathyroid scintigraphy with (99m)Tc-sestaMIBI is a very sensitive technique in the preoperative localization and diagnosis of parathyroid adenoma. However, pitfalls have been reported in patients with thyroid nodules with MIBI uptake or with previous thyroid surgery. To solve this problem, a thyroid scintigraphy with (99m)Tc-pertechnetate is usually performed following the parathyroid study. Occasionally, as in our patient the parathyroid lesion may show high MIBI uptake and delayed washout that interfere with the subsequent thyroid scintigraphy giving the false appearance of a pertechnetate avid lesion. This has been called the «shine through¼ effect. To avoid it, the parathyroid and thyroid scintigraphies can be performed on separate days. We have also found it useful to compare our results with that of ultrasound and fine needle aspiration puncture with measurement of the parathyroid hormone (PTH) and thyroglobulin in the aspirated material.

Identification of several intracellular carbohydrate-degrading activities from the halophilic archaeon Haloferax mediterranei.

Three different amylolytic activities, designated AMY1, AMY2, and AMY3 were detected in the cytoplasm of the extreme halophilic archaeon Haloferax mediterranei grown in a starch containing medium. This organism had also been reported to excrete an alpha-amylase into the external medium in such conditions. The presence of these different enzymes which are also able to degrade starch may be related to the use of the available carbohydrates and maltodextrins, including the products obtained by the action of the extracellular amylase on starch that may be transported to the cytoplasm of the organism. The behavior of these intracellular hydrolytic enzymes on starch is reported here and compared with their extracellular counterpart. Two of these glycosidic activities (AMY1, AMY3) have also been purified and further characterized. As with other halophilic enzymes, they were salt dependent and displayed maximal activity at 3 M NaCl, and 50 degrees C. The purification steps and molecular masses have also been reported. The other activity (AMY2) was also detected in extracts from cells grown in media with glycerol instead of starch and in a yeast extract medium. This enzyme was able to degrade starch yielding small oligosaccharides and displayed similar halophilic behavior with salt requirement in the range 1.5-3 M NaCl.

Composting olive mill pomace and other residues from rural southeastern Spain.

The costly disposal of the semisolid residual pomace generated in the two phase extraction used in modern olive mills is causing serious problems to the small oil producers of rural southeastern Spain. Composting may be a viable alternative since complementary residues are usually available in these areas to prepare an adequate starting mixture. In this work, four different combinations of residues (pomace+rabbit manure, pomace+sheep manure, pomace+rabbit manure+rice straw, pomace+rabbit manure+almond shells) were composted in 3 ton piles aerated by turnings, using technology available to any small community of oil producers. During the four long processes (9-10 months), a steady decrease of organic matter and increases in the concentrations of nutrient and humic substances were observed, together with large increases in pH and salinity which may reduce the agronomic value of the final products.

Calcificaciones intracraneales extensas en el hipoparatiroidismo crónico / Extensive intracranial calcification in chronic hypoparathyroidism

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Alpha-amylase activity from the halophilic archaeon Haloferax mediterranei.

The halophilic archaeon Haloferax mediterranei is able to grow in a minimal medium containing ammonium acetate as a carbon and nitrogen source. When this medium is enriched with starch, alpha-amylase activity is excreted to the medium in low concentration. Here we report methods to concentrate and purify the enzyme. The relative molecular mass of the enzyme, determined by gel filtration, is 50 +/- 4 kDa, and on SDS-PAGE analysis a single band appeared at 58 kDa. These results indicated that the halophilic alpha-amylase is a monomeric enzyme. The enzyme showed a salt requirement for both stability and activity, being stable from 2 to 4 M NaCl, with maximal activity at 3 M NaCl. The enzyme displayed maximal activity at pHs from 7 to 8, and its optimal temperature was in a range from 50 degrees C to 60 degrees C. The results also implicated several prototropic groups in the catalytic reaction.

Organic matter components, aggregate stability and biological activity in a horticultural soil fertilized with different rates of two sewage sludges during ten years.

The effects of the application as fertilizer during ten years of two sewage sludges (aerobically and anaerobically digested, at rates of 400, 800, and 1200 kg of N/ha yr), on the aggregate stability and contents of related organic matter components, microbial biomass and levels of five enzymatic activities (alkaline phosphomonoesterase, phosphodiesterase, urease, arylsulphatase and dehydrogenase) were investigated. The application of both sludges at mid and high rates gave rise to significant increases of organic matter, humified substances and humic acids, but no effects on carbohydrates, microbial gums and aggregate stability were observed. As for biological activity in soils, the high variability of data led to a general absence of statistical significance despite the large differences between treatments observed. Significant increases of phosphodiesterase activity were nevertheless produced by the high rate of aerobic sludge and the mid and high rates of the anaerobic sludge.

Organic matter components and aggregate stability after the application of different amendments to a horticultural soil.

The effects of usual or recommended rates of application of five organic amendments (24 t/ha yr of MSW compost, sewage sludge, and ovine manure, 2.4 t/ha yr of commercial vermicompost, and 100 l/ha yr of a commercial humic acids solution) on the soil contents of organic matter, total humified substances, humic acids, carbohydrates and microbial gums, and the structural stability of aggregates were investigated. Four and five years after the beginning of the experiment, significant increments in most of the parameters studied were found after the application of organic residues, whereas the two commercial amendments did not produce any significant change, suggesting that rates recommended by the producers and imposed by their high prices are too low to be useful. MSW compost yielded the highest increases, even if the amount of organic matter applied as ovine manure was very similar. Organic matter and carbohydrates appeared to be the parameters most closely related to soil aggregate stability.

Naturally occurring neuronal death during postnatal development of the cerebellar nuclei in the rat / Muerte neuronal que ocurre de forma natural durante el desarrollo post-natal de los núcleos cerebelares de la rata

Naturally occurring neuronal death during the postnatal development of the cerebellar nuclei in the rat was analysed using classical cell counts and the labelling of fragmented DNA. Cell counts did not demonstrate a clear decrease in the number of cerebellar nuclei neurons along the first thirty days of postnatal life. However, the TUNEL technique revealed the existence of apoptotic cells with their DNA fragmented from the first to twentieth postnatal days. Apoptotic cells were randomly distributed throughout the cerebellar nuclei with no topographic preference and, in all cases, their numbers were very low, ranging from one to eight labelled cells per nucleus and section. The large amount of target (i.e., granule cells) that cerebellar nuclei neurons have could readily explain the absence of an important decrease in neuronal numbers during the postnatal development of these nuclei. The existence of apoptotic cells could be the expression of the withdrawal of neurons erroneously emplaced during the establishment of the refined topographic pattern of the cerebellar nucleo-cortical and cerebellofugal projections (AU)

Se analizó la muerte neuronal que ocurre de una manera natural durante el desarrollo post-natal de los núcleos cerebelosos de la rata utilizando conteos clásicos y el marcaje de DNA fragmentado. Los conteos no revelaron ningún descenso claro en el número de neuronas de los núcleos cerebelosos a lo largo de los primeros 30 días de vida post-natal. No obstante, la técnica TUNEL sí reveló la existencia de células apoptóticas cuyo DNA estaba fragmentado a partir del día 20 post-natal. Las células apoptóticas estaban distribuidas aleatoriamente por todos los núcleos cerebelosos, no mostrando ninguna preferencia topográfica, y en todos los casos sus números eran muy bajos, oscilando entre una y ocho células marcadas por núcleo y sección. La gran cantidad de diana (es decir, las células granulares) poseída por las neuronas de los núcleos cerebelosos fácilmente podría justificar la ausencia de un descenso importante en los números neuronales durante el desarrollo post-natal de estos núcleos. La existencia de células apoptóticas podría representar la expresión de la retirada de neuronas emplazadas de manera errónea durante el establecimiento del patrón topográfico refinado de las proyecciones cerebelosas núcleo-corticales y cerebelofugales (AU)

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum.

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.

Differences between sporadic and multiple endocrine neoplasia type 2A phaeochromocytoma.

OBJECTIVES: Phaeochromocytoma may be either sporadic or part of a familial cancer syndrome. We have investigated whether there are differences between sporadic and MEN 2A phaeochromocytomas. DESIGN: A retrospective study. We analysed age at presentation, sex, mode of presentation, clinical data, laboratory and imaging techniques, treatment, pathology and follow-up. PATIENTS: Forty-six patients diagnosed with phaeochromocytoma between 1979 and 1995 (23 sporadic and 23 familial) at the Hospital Universitario Virgen de la Arrixaca (Murcia, Spain), a tertiary referral centre. RESULTS: Mean age at presentation was 47 +/- 16 years for sporadic and 38 +/- 11 years for familial phaeochromocytoma (P < 0.05). The most common clinical feature and the presenting feature in cases of isolated phaeochromocytoma was hypertension. Of the MEN 2A patients, 52% were asymptomatic at diagnosis and only 35% presented with hypertension. Levels of adrenaline, noradrenaline, dopamine and metanephrines in a 24-hour urine connection confirmed the diagnosis in 95% of the isolated and 91% of the familial phaeochromocytomas. Computed tomography located 100% of the sporadic and 76% of the familial phaeochromocytomas (P < 0.02). All the MEN 2A phaeochromocytomas were bilateral compared with none of the sporadic phaeochromocytomas (P < 0.001). All the patients with familial phaeochromocytoma underwent bilateral adrenalectomy. The patients with sporadic phaeochromocytoma had the affected gland or the tumour, if extraadrenal, removed. CONCLUSIONS: In comparing sporadic and MEN 2A phaeochromocytoma we found differences in age at presentation, mode of presentation, clinical data, duration of clinical features, imaging findings bilaterality and type of surgical treatment required.

NADP-glutamate dehydrogenase from the halophilic archaeon Haloferax mediterranei: enzyme purification, N-terminal sequence and stability.

An NADP(H)-specific glutamate dehydrogenase of Haloferax mediterranei has been purified to apparent homogeneity and characterised. The purified enzyme was stabilized by glycerol in absence of salt. Glutamate dehydrogenase from Hf. mediterranei is a hexameric enzyme with a native molecular mass of 320 kDa composed of monomers each with a molecular mass of 55 kDa. At pH 8.5 the enzyme has Kms of 0.018, 0.34 and 4.2 mM for NADP+, 2-oxoglutarate and ammonium, respectively. Amino acid composition and sequence of the first 16 residues of the N-terminus have been determined.

NAD-glutamate dehydrogenase from Halobacterium halobium: inhibition and activation by TCA intermediates and amino acids.

A variety of metabolites have been found to elicit a form of inhibition or activation on an NAD-specific glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from Halobacterium halobium. The purified halophilic enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzyme activity. GTP, ATP, ADP and AMP did not affect the enzyme, so these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of the halophilic enzyme. However, the halophilic enzyme was subject to strong inhibition by TCA intermediates. When measuring the initial rate of the reaction, the oxidative deamination of L-glutamate was inhibited by TCA metabolites such as: fumarate, oxalacetate, succinate and malate; by substrate analogues such as: NADP+, D-glutamate and glutarate; and by dicarboxylic compounds such as adipate. On the other hand, all the amino acids tested were activators of this enzyme, except the D-isomer of the substrate L-glutamate that acted as an inhibitor. The relative effectiveness of each inhibitor or activator (Ki or Ka values) was correlated with the dipole moment (mu), HOMO and LUMO molecular orbital energies, optimal distance between two carboxyl groups, and hydrophobicity. Compounds with high dipole moment acted as good activators while compounds with low dipole moment were inhibitors. We have also found that the best activators were amino acids with no polar lateral chain.