Mechanism of activation of cAMP-dependent protein kinase: in Mucor rouxii the apparent specific activity of the cAMP-activated holoenzyme is different than that of its free catalytic subunit

Kinetic constants for peptide phosphorylation by the catalytic subunit of the dimorphic fungus Mucor rouxii protein kinase A were determined using 13 peptides derived from the peptide containing the basic consensus sequence RRASVA, plus kemptide, S6 peptide, and protamine. As a whole, although with a greater Km, the order of preference of the peptides by the M. rouxii catalytic subunit was similar to the one displayed by mammalian protein kinase A. Particularly significant is the replacement of serine by threonine in the basic peptide RRATVA, which impaired its role as a substrate of M. rouxii catalytic subunit. Mucor rouxii protein kinase A is a good model in which to study the mechanism of activation since cAMP alone is not enough to promote activation and dissociation. Four peptides were selected for the study of holoenzyme activation under conditions in which the enzymatic activity was not proportional to the holoenzyme concentration: RRASVA, RRRRASVA, KRRRLSSRA (S6 peptide), and LRRASLG (kemptide); protamine was used as reference. Differential activation degree was observed depending on the peptide used and on cAMP concentration. Ratios of activity between different substrates displayed by the holoenzyme under the above conditions did not reflect the one expected for the free catalytic subunit. The degree of inhibition of the holoenzyme activity by an active peptide derived from the thermostable protein kinase inhibitor was dependent on the substrate used and on the holoenzyme concentration, while it was found to be independent of these two parameters for free catalytic subunit. Polycation modulation of holoenzyme activation by cAMP was also dependent on the polycation itself and on the peptide used as substrate. The observed kinetic differences between holoenzyme and free catalytic subunit were decreased or almost abolished when working at low enzyme or at high cAMP concentrations. Two hypotheses compatible with the results are discussed: substrate participation in the dissociation process and/or holoenzyme activation without dissociation.     

Internal Trehalose Protects Endocytosis from Inhibition by Ethanol in Saccharomyces cerevisiae

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.     

Latitudinal variation in parental energy expenditure during brood rearing in the great tit

The variation in time and energy allocation of female great tits, Parus major, was studied in five different European populations across a latitudinal gradient. Daily energy expenditure (DEE) was measured in females tending 12-day-old broods. The number of daylight hours used by the parents to collect food for the brood increased with latitude, while DEE and feeding rate per brood tended to level off with latitude. Individual variation in DEE could be explained by variation in ambient temperature (–), the duration of activity period (+) and area, but not by brood size, female body mass, brood mass or feeding rate. When the effect of ambient temperature and the duration of the activity period on the day of energy expenditure measurements were controlled for, female DEE still tended to level off with latitude. Temperature and activity alone can thus not explain the observed pattern. The present study suggests that parents at southern latitudes may be under a time constraint and do not increase energy expenditure because they have no more daylight hours available for foraging, while birds at northern latitudes may be under an energy constraint because they do not make full use of the long daylight period available. Received: 25 May 1999 / Accepted: 08 September 1999     

Effects of meteorological parameters and plantdistribution on Chenopodiaceae-Amaranthaceae, Quercus and Olea airborne pollen concentrationsin the atmosphere of Cartagena (Spain)

We have studied the effects of various meteorologicalparameters and plant distribution on the airborneconcentrations of three different pollen types:Chenopodiaceae-Amaranthaceae, Quercus and Olea, in the city of Cartagena, situated in SouthwestEurope. The aerobiological study has been carried outfrom March 1993 to March 1997, using a Hirst-typeLanzoni 2000 trap, situated on the roof of the citytrain station. Meteorological data were supplied bythe Environmental Service of Cartagena's Town Hall.Daily pollen concentrations were standardised.Correlation analysis showed the importance ofmeteorological data on airborne pollen concentrations.On the other hand, ANOVA analyses depicted theinfluence of vegetation distribution in relation tosampler location and wind directions. Finally,multiple regression analyses allowed us to developcorrelation models for the three different pollentypes considered, accounting for 32%(Chenopodiaceae-Amaranthaceae), 19% (Quercus),and 26% (Olea) of the variation in the airbornepollen concentration.     

Large-scale T-DNA mutagenesis in Arabidopsis for functional genomic analysis

In planta Agrobacterium-mediated transformation combined with a soil-based herbicide selection for transgenic plants was used to recover large numbers of transgenic Arabidopsis plants for functional genomic studies. A tissue-culture-free system for generating transgenic plants was achieved by infiltrating Arabidopsis plants with Agrobacterium tumefaciens harboring a binary T-DNA vector containing the phosphinothricin acetyltransferase gene from Streptomyces hygroscopicus, and by selecting transgenic Arabidopsis growing in soil by foliar application of the herbicide Finale (phosphinothricin). Analysis of herbicide-resistant plants indicated that all were transgenic and that the T-DNA transformation process occurred late during flower development, resulting in a preponderance of independently derived T-DNA insertions. T-DNA insertions were usually integrated in a concatenated, rearranged form, and using linkage analysis, we estimated that T1 plants carried between one and five T-DNA loci. Using pooling strategies, both DNA and seed pools were generated from about 38,000 Arabidopsis plants representing over 115,000 independent T-DNA insertions. We show the utility of these transgenic lines for identifying insertion mutations using gene sequence and PCR-based screening. Electronic Publication     

High prevalence of Schistosoma japonicum by perfusion in naturally exposed water buffalo in a region of the Philippines endemic for human schistosomiasis

In the past decade, ecological surveys emphasized rats and dogs as the most significant animal reservoirs for Schistosoma japonicum (S.j) in the Philippines. However, recent studies demonstrated 51–91% prevalence of schistosomiasis among water buffalo using qPCR in the Sj endemic regions in the Philippines. In order to resolve the inconsistency of reported surveys regarding Sj endemicity among carabao, a domestic water buffalo that is the most important draught animal, we introduced 42 schistosome negative water buffalo to Macanip, Jaro municipality, Leyte, the Philippines, a subsistence rice-farming village that has been the focus of schistosomiasis japonica studies of our group for the past 20 years. We conducted perfusion to the remaining 34 buffalo that survived 10 months of nature exposure and Typhoon Haiyan. Thirty-three water buffalo were found to be positive with at least 1 pair of worms from the mesenteric vein. The infection rate is 97%, with the worm burden of 94 (95% confidence interval, 49–138 worms) worms. To our knowledge, this is the first report about S. japonicum worm burden in naturally infected water buffalo in the Philippines. The fact that with less than one-year of exposure, in this human schistosomiasis endemic area, only 1 out of 34 water buffalo was uninfected is striking. Urgent attention is needed for a cost-effective technique for monitoring Sj infection in animals and humans. Meanwhile, intervention implementation, including water buffalo treatment and vaccination, should be taken into consideration.     

FOXE1 polymorphisms and chronic exposure to nitrates in drinking water cause metabolic dysfunction,thyroid abnormalities,and genotoxic damage in women

Nitrates in drinking water has been associated to adverse health effects, including changes in glucose and lipid levels, thyroid hormone imbalance and adverse reproductive effects. We analyzed metabolic and thyroid hormone alterations and genotoxic damage in women with chronic exposure to nitrates in drinking water. The concentration of nitrates in drinking water was quantified and according to this parameter, participants were divided into three exposure scenarios. Blood and urine samples were collected from 420 women living in Durango, Mexico and biomarkers were determined. We found nitrates concentrations in drinking water above the permissible limit (>50 mg/L), and an increase in the percentage of methemoglobin (p=0.0001), nitrite in blood plasma and urine (p=0.0001), glucose (p=0.0001), total cholesterol (p=0.001), LDL (p=0.001) and triglycerides (p=0.0001). We also found alterations in TSH (p=0.01), fT3 (p=0.0003), T4T (p=0.01) and fT4 (p=0.0004) hormones. Frequency of subclinical hypothyroidism was 8.33%; differences in FOXE1 (rs965513, rs1867277) genotypes distribution were found and both polymorphisms were associated with a decrease in TSH. A high percentage of micronucleus in binucleate lymphocyte cells was found (35%, p=0.0001). In conclusion, the chronic exposure to nitrates in water for human consumption caused metabolic and hormonal alterations and genotoxic damage in women.     

Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease.     

Reduction of the metallochromic indicators arsenazo III and antipyrylazo III to their free radical metabolites by cytoplasmic enzymes

At a concentration much lower than that usually employed for measuring cytosolic ionized Ca²⁺ concentrations, arsenazo III underwent a one-electron reduction by rat liver cytosolic fraction or a hypoxanthinexanthine oxidase system to produce an azo anion radical metabolite. NADH, NADPH, N¹-methylnicotinamide, hypoxanthine, and xanthine, in that order, could serve as a source of reducing equivalents for the production of this free radical by the cytosolic fraction. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated O₂ consumption were enhanced by calcium and magnesium. Antipyrylazo III was ineffective in increasing O₂ consumption by rat liver cytosolic fraction and gave a much weaker ESR signal of an azo anion radical with both the liver cytosolic fraction, in the presence of NADH, and the hypoxanthine-xanthine oxidase system.