A new spectrophotometric assay for dopachrome tautomerase

The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nm on the basis of its ability to catalyze dopachrome decoloration. This method presents two major problems, derived from the instability of the substrate (dopachrome): (1) dopachrome must be prepared immediately before use, and (2) the rate of dopachrome decoloration in the absence of the enzyme is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In order to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrome from L-dopa by sodium periodate oxidation; (2) a spectrophotometric method in the UV region, at 308 nm, based on following the absorbance increase due to the enzyme-specific tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid as opposed to the absorbance decrease due to the spontaneous decarboxylative transformation of dopachrome into 5,6-dihydroxyindole. The advantages of these methods as compared to the previously used procedures are discussed.     

Production of hydrogen peroxide by aryl-alcohol oxidase from the ligninolytic fungusPleurotus eryngii

Summary Production of extracellular hydrogen peroxide by fungal oxidases is been investigated as a requirement for lignin degradation. Aryl-alcohol oxidase activity is described in extracellular liquid and mycelium ofPleurotus eryngii and studied under non-limiting nitrogen conditions. This aryl-alcohol oxidase catalyses conversion of primary aromatic alcohols to the corresponding aldehydes and H₂O₂, showing no activity with aliphatic and secondary aromatic alcohols. The enzyme is stable at pH 4.0–9.0, has maximal activity at 45°–50°C and pH 6.0–6.5, is inhibited by Ag⁺, Pb²⁺ and NaN₃, and has aK _m of 1.2 mM using veratryl alcohol as substrate. A single protein band with aryl-alcohol oxidase activity was found in zymograms of extracellular and intracellular crude enzyme preparations fromP. eryngii.     

Substrate-dependent degradation patterns in the decay of wheat straw and beech wood by ligninolytic fungi

Summary The ability of 45 fungal strains to degrade wheat straw and beech wood was studied. Degradation patterns were defined in terms of chemical evolution of substrates and changes in lignin and polysaccharides. Trametes versicolor produced an important degradation of lignin and increased substrate digestibility, but it caused high weight losses and gave rise to similar decay patterns on both substrates. A preferential degradation of lignin was produced during straw transformation by Pleurotus eryngii. The increase of soluble lignin and decreases of lignin content and H/C ratio defined the degradation tendency after principal component analysis. The cation exchange capacity and water and alkali solubility presented the highest loading factors for the characterization of fungal transformation of beech wood. Offprint requests to: A. T. Martínez     

Statistical analysis for the spatial validity of a model to forecast the daily number of forest fires

In earlier papers a qualitative and quantitative model was developed for predicting the number of forest fires occurring per day. This model permits the forecast at 00.00 hours Universal Time Convention (UTC) of any day (d), the number of forest fires per day for a range of several days (d tod+5) over a particular region. Input data are the number of forest fires in the region during two preceding days (d–2 andd–1) and the type of day (real and evaluated from radiosonde ford–2,d–1,d and predicted from meteorological medium-range forecasts, i.e. of European Centre, ford+1,d+2,d+3,d+4 andd+5. As this model requires data obtained by radiosonde, particularly temperatures and geopotentials at 850 and 700 hPa and dew points (or specific humidity) at 850 hPa, this study investigates the spatial validity of the model in relation to the distance from the radiosonde station (RS). The highest quality forecast is obtained for the region immediately surrounding the RS, and diminishes with increasing distance from it, this being due to the data obtained from the RS not being representative of the atmospheric column over the region. Hence, the derivation of the critical distance for a particular quality level of measurement. Conversely, fixed quality level implies a specific separation between RS and the region for the prediction, with a higher predictive quality implying a shorter distance.     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Sex steroid effects on the development and functioning of the growth hormone axis

Summary 1. The secretory pattern of growth hormone (GH) is sexually dimorphic in the adult rat. However, this difference between the sexes does not become apparent until after the onset of puberty, suggesting that pubertal sex steroids play an important role in the manifestation of this phenomenon.2. We have addressed the question as to whether there exists a sexual dimorphism in the hypothalamic neuropeptides that regulate GH release from the anterior pituitary,i.e., somatostatin (SS) and growth hormone-releasing hormone (GHRH). In addition, we have investigated whether the developmental changes in the GH secretory pattern are correlated with changes in these neuropeptides. The effect of testosterone treatment on SS and GHRH neurons during both the neonatal period and adulthood have also been studied.3. We have found that the synthetic capacity, as reflected in relative messenger RNA (mRNA) levels, of both SS and GHRH neurons changes throughout development in both male and female rats. These mRNA levels are sexually dimorphic at certain times during maturation and can be modulated by changes in testosterone levels, suggesting that sex steroid modulation of these two neuropeptide systems could at least partially account for the sexual dimorphism seen in the adult GH secretory pattern.4. The neonatal steroid environment has also been suggested to be involved in the generation of the final adult GH secretory pattern, although the mechanisms underlying this effect are even less well understood. In support of the hypothesis that the neonatal steroid environment plays an important role in organizing the GH axis, we have found that the number of GHRH neurons in the adult brain, as well as their sensitivity to adult steroids, is modulated by neotatal testosterone treatment. The number of SS neurons in the periventricular and paraventricular nuclei were not modulated by neonatal steroids; however, the synthetic capacity of these neurons does appear to be influenced by the neonatal steroid environment.5. These studies suggest that both the neonatal and adult sex steroid environments influence the adult GH secretory pattern by modulating GHRH and SS neurons.     

Sex masks: The double life of female commercial sex workers in Mexico City

The central topic of the article is the divided world of female commercial sex workers (FCSW) in Mexico City. Fourteen focus group sessions were conducted with 133 FCSW from varying socio-economic levels and types of work site, as well as seven individual interviews.FCSW live in a constant double bind, as mother and prostitute, and come into daily contact with society's double standard for women. Reactions include justifying sex work as a better paying employment opportunity for women, as a necessary evil, and as a type of social service, while at the same time hiding their profession from their families. FCSW also live out an archetypal female ambivalence, their selves divided between the mother/saint and the traitor/prostitute.This article defines elements which should be taken into account in culturally appropriate programs for prevention of HIV/AIDS transmission, especially the importance which FCSW give to their role as mothers and promotion of the condom as a physical and symbolic barrier between professional and private life.     

In vitro culture selection increases glyphosate tolerance in barley

In vitro culture of barley calluses has been used to produce plants with increased glyphosate tolerance. Calluses from immature embryos of barleyHordeum vulgare L. (Jeff) were cultured on Murashige and Skoog medium with 10^-6, 10^-5, 10^-4, 5×10^-4, 10^-3, or 10^-2M glyphosate for one, four or thirty months. Plants were regenerated from calluses maintained in glyphosate medium at 10^-6, 10^-5 or 10^-4M for four months, at 10^-5 or 5×10^-4M for one month and at 10^-5M for thirty months. The progeny of each regenerated plant was analyzed for response to glyphosate. Some progenies showed increased tolerance to glyphosate.     

Mixed connective tissue disease. A clinico-serological study of 17 cases

Mixed connective tissue disease (MCTD) was described as a distinct clinical syndrome in 1972. Since then many cases have been reported in the literature worldwide. In this study we present our experience with a group of 17 Mexican patients with this syndrome, and we analyze their clinical and serological features, as well as the causes of death in these patients. The patients are Mexican mestizos living in Guadalajara and most of them have been followed-up at Hospital General de Occidente for a period of 1–10 years. The female/male ratio was 16:1, and their age ranged from 14–55 years with a mean of 29 years. The disease duration has ranged from 1–17 years, with a mean of 6 years. Among the clinical manifestations we have found a high frequency of lymphadenopathy when compared with published series (13/17 or 76%), and the laboratory findings in our patients included a very high polyclonal increase of gammaglobulins (93%), lymphopenia (76%), direct immunofluorescence speckled nuclear epidermal deposits in skin biopsies (75%) and positive rheumatoid factor (65%). Other clinical and serological features were similar to those reported in other series of patients with MCTD. Six of the 17 patients have died (35%), and in 3 of them (17.5%) the cause of death was due to an infectious disease that suddenly presented, and apparently was not related to a concomitant high dose of steroids or malnutrition in the patients. It seems that in addition to the already well known autoimmune abnormalities that occur in MCTD, there are other features like the presence of lymphadenopathy, the high polyclonal increase of gammaglobulins, and the lymphopenia, that reflect the profound disturbance of the immune system in this syndrome, possibly contributing to the sudden appearance of a severe infectious disease in some of our patients.Abbreviations ANA antinuclear antibody - CIE counterimmunoelectrophoresis - MCTD mixed connective tissue disease - PHA passive hemagglutination - PM polymyositis - RF rheumatoid factor - SLE systemic lupus erythematosus - SS systemic sclerosis (SS)