Melanin is crucial for growth of the black yeast Hortaea werneckii in its natural hypersaline environment

Melanin has an important role in the ability of fungi to survive extreme conditions, like the high NaCl concentrations that are typical of hypersaline environments. The black fungus Hortaea werneckii that has been isolated from such environments has 1,8-dihydroxynaphthalene-melanin incorporated into the cell wall, which minimises the loss of glycerol at low NaCl concentrations. To further explore the role of melanin in the extremely halotolerant character of H. werneckii, we studied the effects of several melanin biosynthesis inhibitors on its growth, pigmentation and cell morphology. The most potent inhibitors were a 2,3-dihydrobenzofuran derivative and tricyclazole, which restricted the growth of H. werneckii on high-salinity media, as shown by growth curves and plate-drop assays. These inhibitors promoted release of the pigments from the H. werneckii cell surface and changed the medium colour. Inhibitor-treated H. werneckii cells exposed to high salinity showed both decreased and increased cell lengths. We speculate that this absence of melanin perturbs the integrity of the cell wall in H. werneckii, which affects its cell division and exposes it to the harmful effects of high NaCl concentrations. Surprisingly, melanin had no effect on H. werneckii survival under H₂O₂ oxidative stress.     

Joint effect of G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism on the risk of premature coronary artery disease

The study sought an association between the G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism and premature coronary artery disease (CAD), and the interactive effect on CAD risk between the G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism as well as between tested polymorphisms and traditional risk factors. 167 patients with CAD younger than 55 years were compared with 132 healthy subjects. The frequency of factor V point mutation was 7.8 % among Slovene patients with premature CAD, and 4.5 % among controls. No association was found between either the factor V point mutation (AG genotype) or M1M1 genotype of factor VII Arg/Gln(353) gene polymorphism and the risk of CAD in Slovenia using univariate analysis (factor V point mutation: OR = 1.8, 95% CI = 0.7-4.9; p = 0.25; factor VII Arg/Gln(353) gene polymorphism: OR = 1, 95 % CI = 0.6-1.7; p = 0.9). However, a joint effect on the risk of CAD was found between factor V point mutation (AG genotype) and M1M1 genotype (OR = 3.6, 95 % CI = 1-12.9; p = 0.03). Additionally, an interactive effect on CAD risk was found between AG genotype and metabolic risk factors (OR = 3.8, 95% CI = 1.1-13.6; p = 0.03). In conclusion, we provide evidence for a joint effect on CAD risk between G1691A factor V point mutation and factor VII Arg/Gln(353) gene polymorphism as well as between factor V point mutation and metabolic risk factors.     

Genomic DNA sequence of Rhesus (M. mulatta) cystic fibrosis (CFTR) gene

Cystic fibrosis is a common human genetic disease caused by mutations in CFTR, a gene that codes for a chloride channel that is regulated by phosphorylation and cytosolic nucleotides. As part of a program to discover natural animal models for human genetic diseases, we have determined the genomic sequence of CFTR in the Rhesus monkey, Macaca mulatta. The coding region of rhesus CFTR is 98.3% identical to human CFTR at the nucleotide level and 98.2% identical and 99.7% similar at the amino acid level. Partial sequences of flanking introns (5582 base pair positions analyzed) revealed 91.1% identity with human introns. Relative to rhesus intronic sequence, the human sequences had 27 insertions and 22 deletions. Primer sequences for amplification of rhesus genomic CFTR sequences are provided. The accession number is AF013753 (all 27 exons and some flanking intronic sequence). Received: 27 August 1992 / Accepted: 5 December 1997     

Metal binding of metallothioneins in human astrocytomas (U87 MG,IPDDC-2A)

Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl₂ (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.     

Identification and characterization of putative osmosensors, HwSho1A and HwSho1B, from the extremely halotolerant black yeast Hortaea werneckii

In Saccharomyces cerevisiae, the Sho1 protein is one of two potential osmosensors that can activate the kinase cascade of the HOG pathway in response to increased extracellular osmolarity. Two novel SHO1-like genes, HwSHO1A and HwSHO1B, have been cloned from the saltern-inhabiting, extremely halotolerant black yeast Hortaea werneckii. The HwSho1 protein isoforms are 93.8% identical in their amino-acid sequences, and have a conserved SH3 domain. When the HwSHO1 genes were transferred into S. cerevisae cells lacking the SHO1 gene, both of the HwSho1 isoforms fully complemented the function of the native S. cerevisiae Sho1 protein. Through microscopic and biochemical validation, we demonstrate that in S. cerevisiae, both of the HwSho1 proteins have characteristic subcellular localizations similar to the S. cerevisiae Sho1 protein, and they can both activate the HOG pathway under conditions of osmotic stress. To a lower extent, crosstalk to the mating pathway expressing HwSho1 proteins is conserved in the PBS2 deleted S. cerevisiae strain. These data show that the HwSho1 proteins from H. werneckii are true functional homologs of the Sho1 protein of S. cerevisiae.     

In silico fragment-based discovery of indolin-2-one analogues as potent DNA gyrase inhibitors

We describe here the fragment-based design of potent DNA gyrase inhibitors. Using the tools of virtual screening and NMR spectroscopy we identified the binding of two low-molecular weight fragments (2-aminobenzimidazole and indolin-2-one) to the 24kDa N-terminal fragment of DNA gyrase B. Further in silico optimization of indolin-2-one led to the discovery of potent DNA gyrase inhibitors.     

Recent vegetation history of the North Adriatic grasslands: Expansion and decay of an anthropogenic habitat

A summary pollen diagram and old cartographic materials were used to reconstruct the recent vegetation history and trends of temporal dynamics of the North Adriatic Karst grasslands. Before the Roman period some evidence about grassland vegetation exists from Neolithic settlements, but deforestation, detectable on the landscape level because of pollen, started 2500-2000 years BP. A large-scale anthropogenically-driven process of clearance started in the Roman period as is reflected in the increasing pollen of grasses (Poaceae) and other herbaceous species (Apiaceae). The presence of pioneer trees (Quercus, Ostrya), juniper and grasses during 1000-400 years BP suggests that cycling processes of cultivation (strong grazing pressure), abandonment and re-cultivation were present. A 250-year-old map shows a peak of probable deforestation, where grasslands have 3.2 times larger surface area than on a recent land-use map in a 665.8 km² large pilot area. More than 60% of grasslands changed into forest since then. On a sub-plot area of 626 ha (1% of the pilot area), where habitat mappings were performed, only 12.8% were still grasslands without tall herb invasions or scrub encroachments. Another 27.1% were grasslands with early signs of reforestation. The traditionally open landscape of the North Adriatic Karst was able to recover to forest due to almost total abandonment and sufficient mesic climate conditions.