Phosphorylation of high-mobility group protein A2 by Nek2 kinase during the first meiotic division in mouse spermatocytes

The mitogen-activated protein kinase (MAPK) pathway is required for maintaining the chromatin condensed during the two meiotic divisions and to avoid a second round of DNA duplication. However, molecular targets of the MAPK pathway on chromatin have not yet been identified. Here, we show that the architectural chromatin protein HMGA2 is highly expressed in male meiotic cells. Furthermore, Nek2, a serine-threonine kinase activated by the MAPK pathway in mouse pachytene spermatocytes, directly interacts with HMGA2 in vitro and in mouse spermatocytes. The interaction does not depend on the activity of Nek2 and seems constitutive. On progression from pachytene to metaphase, Nek2 is activated and HMGA2 is phosphorylated in an MAPK-dependent manner. We also show that Nek2 phosphorylates in vitro HMGA2 and that this phosphorylation decreases the affinity of HMGA2 for DNA and might favor its release from the chromatin. Indeed, we find that most HMGA2 associates with chromatin in mouse pachytene spermatocytes, whereas it is excluded from the chromatin upon the G2/M progression. Because hmga2-/- mice are sterile and show a dramatic impairment of spermatogenesis, it is possible that the functional interaction between HMGA2 and Nek2 plays a crucial role in the correct process of chromatin condensation in meiosis.     

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.     

Amino Acid Sequence and Molecular Weight of Native NADP Malate Dehydrogenase from the C(4) Plant Zea mays

N-terminus amino acid analysis of purified corn (Zea mays) NADP malate dehydrogenase showed that the mature protein begins at serine-41 of the preprotein sequence and not threonine-58 as previously concluded; therefore, the transit peptide consists of 40 amino acids. The theoretical molecular weight of the mature subunit protein (392 amino acids) is 42,564, agreeing with an experimental value of about 43,000. The molecular weight of the native unactivated (dark form) and activated (light form) of NADP malate dehydrogenase, determined by analytical ultracentrifugation analysis, was about 84,000, indicating that both forms are dimers. However, conventional and high performance liquid chromatography gel filtration procedures indicated apparent molecular weights of about 110,000 to 120,000 for the unactivated native enzyme and about 143,000 to 150,000 for the active enzyme; in these cases, the molecular weight may be overestimated due to the effect of an unusual molecular conformation on the mobility of the enzyme.     

Adenosine deaminase polymorphism in sardinia

Summary Red blood cell adenosine deaminase and G-6-PD polymorphisms have been studied in the populations of 17 Sardinian villages located at various altitudes. A total of 1615 individuals of both sexes, with age between 7 and 14 years were examined.The negative relationship between Gd^Med gene frequency and altitude was confirmed.Adenosine deaminase polymorphism showed a considerable variability among the villages examined: ADA² frequency ranged from 0.025 to 0.106. The ADA² frequency was negatively related (p< 0.05) with Gd^Med gene frequency.

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Zusammenfassung Die Erythrocyten-Adenosin-Desaminase und Glucose-6-Phosphat-Dehydrogenase-Polymorphismen sind in Stichproben aus der Bevölkerung von 17 Dörfern auf Sardinien untersucht worden. Diese sind in verschiedener Höhe über dem Meer im zentralen Teil der Insel gelegen.Die negative Häufigkeitsbeziehung zwischen der Genfrequenz Gd^Med und der Höhenlage der Dörfer konnte bestätigt werden.Der Adenosin-Desaminase-Polymorphismus zeigt eine beträchtliche Variabilität zwischen den verschiedenen Dorfpopulationen. Die ADA²-Frequenz liegt zwischen 0,025 und 0,106. Die ADA²-Frequenz steht in negativer Beziehung (p< 0,05) zur Gd^Med-Frequenz.

     

Growth ofCandida albicans in a minimal synthetic medium without biotin

Summary Growth ofCandida albicans strain B 311-10 was observed in a minimal synthetic biotin-free medium, using different glucose concentrations, during the first 30 hours of its development at 28 °C. The yeast's growth was observed spectrophotometrically at 675 nm reading its optical density every hour. The minimal medium of Shepherdet al. [12], with glucose (15 g/L) and biotin was modified: this vitamin was eliminated and the concentration of glucose was gradually lowered to 0.5 g/L. At 5 g/L of glucose and without biotin very good growth was obtained. Based on our results during the first 30 hours of growth, biotin has no influence on the yeast's growth. This medium would be useful for the study of the physiology ofC. albicans during the first period of its development.     

Magnetic Resonance Imaging Determined Visceral Fat Reduction Associates with Enhanced IL-10 Plasma Levels in Calorie Restricted Obese Subjects

Background

Obesity is characterized by a low grade chronic inflammation state. Indeed circulating pro-inflammatory cytokines, such as TNF-α and IL-6, are elevated in obese subjects, while anti-inflammatory cytokines, such as IL-10, appear to be reduced. Cytokines profile improves after weight loss, but how visceral or subcutaneous fat loss respectively affect pro- or anti-inflammatory cytokines plasma levels has not been precisely assessed. Therefore in the present study we correlated changes in circulating cytokine profile with quantitative changes in visceral and subcutaneous adipose tissue depots measured by an ad hoc Magnetic Resonance Imaging (MRI) protocol before and after weight loss.

Materials and Methods

In 14 obese subjects, MRI determination of visceral and subcutaneous fat and plasma glucose, insulin, TNF-α IL-6, and IL-10 measurements were performed before and after a caloric restriction induced weight loss of at least 5% of the original body weight.

Results

Weight loss improved insulin sensitivity (QUICKI Index: 0.35±0.03 vs 0.37±0.04; P<0.05), increased IL-10 (3.4±1.9 vs 4.6±1.0 pg/mL; P<0.03), and reduced TNF-α and IL-6 plasma levels (2.5±1.3 vs 1.6±1.5 pg/mL, P<0.0015, 2.3±0.4 vs 1.6±0.6 pg/mL, P<0.02 respectively). A significant correlation was observed between the amount of visceral fat loss and the percentage reduction in both TNF-α (r = 0.56, p<0.05) and IL-6 (r = 0.19 p<0.05) plasma levels. In a multiple regression analysis, the amount of visceral fat loss independently correlated with the increase in IL-10 plasma levels.

Conclusion

The reduction in visceral adipose tissue is the main driver of the improved inflammatory profile induced by weight loss.     

Comparative next-generation mapping of the Phytophthora infestans resistance gene Rpi-dlc2 in a European accession of Solanum dulcamara

Phytophthora infestans, the causal agent of late blight, remains the main threat to potato production worldwide. Screening of 19 accessions of Solanum dulcamara with P. infestans isolate Ipo82001 in detached leaf assays revealed strong resistance in an individual belonging to accession A54750069-1. This plant was crossed with a susceptible genotype, and an F₁ population consisting of 63 individuals was obtained. This population segregated for resistance in 1:1 ratio, both in detached leaf assays and in an open-field experiment. Presence of the formerly mapped Rpi-dlc1 gene as the cause of the observed segregating resistance could be excluded. Subsequently, AFLP analyses using 128 primer combinations enabled identification of five markers linked to a novel resistance gene named Rpi-dlc2. AFLP markers did not show sequence similarity to the tomato and potato genomes, hampering comparative genetic positioning of the gene. For this reason we used next-generation mapping (NGM), an approach that exploits direct sequencing of DNA (in our case: cDNA) pools from bulked segregants to calculate the genetic distance between SNPs and the locus of interest. Plotting of these genetic distances on the tomato and potato genetic map and subsequent PCR-based marker analysis positioned the gene on chromosome 10, in a region overlapping with the Rpi-ber/ber1 and -ber2 loci from S. berthaultii. Pyramiding of Rpi-dlc2 and Rpi-dlc1 significantly increased resistance to P. infestans, compared with individuals containing only one of the genes, showing the usefulness of this strategy to enhance resistance against Phytophthora.     

Puccinellia festucaeformis (Host) Parl.: Germinazione e crescita iniziale in funzione della salinità del substrato

Abstract

Puccinellia festucaeformis (Host) Parl.: germination and early growth on different salt substrates. Germination behaviour of Puccinellia festucaeformis seeds and early growth of seedlings at different experimental conditions was analysed. The following growth substrates were utilized: NaCl, KCl, KNO₃, MgCl₂, MgSO₄, Na₂SO₄, NaNO₃, CaCl₂ at the decreasing concentrations of 0.50, 0.25, 0.12, 0.06M. Caryopses were allowed to imbibe and grow at alternating temperatures (10°-20°C or 20°-30°C) in the dark for 3 days. Seedling were grown for 15 days, at controlled light and temperature conditions, in the same nutrient substrates as those used for the germination experiments.

The germination experiments showed a high tolerance to salts up to 0.25M solution and for the whole range of MgSO₄ concentrations. High growth temperatures increased the depressive effects of salt concentrations. Seedling growth was highly reduced when salt concentration was higher than 0.12M. High salt tolerance - maximum shoot and root growth - was showed by seedling allowed to grow on 0.50M MgSO₄.

Germination and growth condition of Puccinellia festucaeformis is discussed in relation to the ecological features of this species and to its possible importance as bioindicator of MgSO₄ rich natural substrates.