Specific and selective peptide-membrane interactions revealed using quartz crystal microbalance

The skin secretions of Australian tree frogs are rich in peptides with potential antimicrobial activity. They interrupt bacterial cell membranes, although precisely how and whether all peptides have the same mechanism is not known. The interactions of three of these peptides—aurein 1.2, maculatin 1.1, and caerin 1.1 with supported phospholipid bilayers—are examined here using quartz crystal microbalance and atomic force microscopy. These approaches enabled us to reveal variations in material structure and density as a function of distance from the sensor surface when comparing mass sensorgrams over a range of harmonics of the natural resonance of the sensor crystal and hence obtain for the first time to our knowledge a mechanistic assessment of membrane disruption. We found that caerin inserted into the bilayer in a transmembrane manner, regardless of concentration and phospholipid composition consistent with a pore-forming mechanism. In contrast, maculatin and aurein interacted with membranes in a concentration-dependent manner. At low concentrations (<5 μM), maculatin exhibited transmembrane incorporation whereas aurein was limited to surface association. Upon reaching a threshold value of concentration, both peptides lysed the membrane. In the case of maculatin, the lysis progressed in a slow, concentration-dependent manner, forming mixed micelles, as shown by atomic force microscopy imaging. Aurein-induced lysis proceeded to a sudden disruption, which is consistent with the “carpet” mechanism. Both maculatin and aurein exhibit specificity toward phospholipids and thus have potential as candidates as antimicrobial drugs.     

Antioxidant defence enzyme activities in hepatopancreas, gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube

The aim of our study was to determine the activity of antioxidant defence (AD) enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the phase II biotransformation enzyme glutathione-S-transferase (GST) in the hepatopancreas, the gills and muscle of Spiny cheek crayfish (Orconectes limosus) from the River Danube and to compare tissue specificities of investigated enzymes. Our results indicated that both specific and total SOD activities in the hepatopancreas were lower compared to the gills and muscle. Total SOD activity in the gills was lower with respect to that in muscle. CAT and GSH-Px (both specific and total) activities were higher in the hepatopancreas compared to those in the gills and muscle. In the gills the specific and total GR activities were higher than in the hepatopancreas and muscle. The specific and total GST activities were higher in the hepatopancreas compared with the gills and muscle. Our study represents the first comprehensive report of AD enzymes in tissues of O. limosus caught in the River Danube. The noted tissue distributions of the investigated AD enzyme activities most likely reflected different metabolic activities and different responses to environmental conditions in the examined tissues.     

The chemical composition of the meat of fat dormice (<Emphasis Type="Italic">Glis glis</Emphasis> L.)

The fat dormouse (Glis glis) is a traditional game species in the Republic of Croatia. Although today the fat dormouse is not frequently caught as game, it is still a source of animal protein in human nutrition in certain rural areas of Croatia. In this paper the chemical analysis of fat dormouse meat is presented. The average values for the quantity of water, fat, protein and ash in dormouse meat are comparable with the chemical composition of the meat of rabbits and brown hares, except for the important fact that rabbit and hare meat contains a greater quantity of fat on average. According to our results, the meat of fat dormice can be categorised as dietary food, characterised by a small percentage of fat (mean 2.83%) and a high amount of protein (mean 21.01%).     

Secondary metabolism in Fusarium fujikuroi: strategies to unravel the function of biosynthetic pathways

The fungus Fusarium fujikuroi causes bakanae disease of rice due to its ability to produce the plant hormones, the gibberellins. The fungus is also known for producing harmful mycotoxins (e.g., fusaric acid and fusarins) and pigments (e.g., bikaverin and fusarubins). However, for a long time, most of these well-known products could not be linked to biosynthetic gene clusters. Recent genome sequencing has revealed altogether 47 putative gene clusters. Most of them were orphan clusters for which the encoded natural product(s) were unknown. In this review, we describe the current status of our research on identification and functional characterizations of novel secondary metabolite gene clusters. We present several examples where linking known metabolites to the respective biosynthetic genes has been achieved and describe recent strategies and methods to access new natural products, e.g., by genetic manipulation of pathway-specific or global transcritption factors. In addition, we demonstrate that deletion and over-expression of histone-modifying genes is a powerful tool to activate silent gene clusters and to discover their products.

     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

The kinesin of the flagellum attachment zone in Leishmania is required for cell morphogenesis,cell division and virulence in the mammalian host

Leishmania parasites possess a unique and complex cytoskeletal structure termed flagellum attachment zone (FAZ) connecting the base of the flagellum to one side of the flagellar pocket (FP), an invagination of the cell body membrane and the sole site for endocytosis and exocytosis. This structure is involved in FP architecture and cell morphogenesis, but its precise role and molecular composition remain enigmatic. Here, we characterized Leishmania FAZ7, the only known FAZ protein containing a kinesin motor domain, and part of a clade of trypanosomatid-specific kinesins with unknown functions. The two paralogs of FAZ7, FAZ7A and FAZ7B, display different localizations and functions. FAZ7A localizes at the basal body, while FAZ7B localizes at the distal part of the FP, where the FAZ structure is present in Leishmania. While null mutants of FAZ7A displayed normal growth rates, the deletion of FAZ7B impaired cell growth in both promastigotes and amastigotes of Leishmania. The kinesin activity is crucial for its function. Deletion of FAZ7B resulted in altered cell division, cell morphogenesis (including flagellum length), and FP structure and function. Furthermore, knocking out FAZ7B induced a mis-localization of two of the FAZ proteins, and disrupted the molecular organization of the FP collar, affecting the localization of its components. Loss of the kinesin FAZ7B has important consequences in the insect vector and mammalian host by reducing proliferation in the sand fly and pathogenicity in mice. Our findings reveal the pivotal role of the only FAZ kinesin as part of the factors important for a successful life cycle of Leishmania.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

A human origin type II strain of Toxoplasma gondii causing severe encephalitis in mice

Despite its capacity for sexual reproduction and global distribution, Toxoplasma gondii has a highly clonal structure, with the majority of isolates belonging to three distinct clonal types. Congenital toxoplasmosis has been associated with type I and type II strains. We here present the first characterization of a T. gondii strain (BGD1) from South-East Europe, isolated from the umbilical blood of a 24-week-old fetus in Serbia. Genotyping, performed by PCR-RFLP using a set of nested PCR markers (5'SAG2, 3'SAG2, BTUB, SAG3, and GRA6), showed that the BGD1 strain possessed a type II genotype. The cytokine patterns in Swiss-Webster mice inoculated with brain cysts of BGD1 and the prototype type II ME49 strain were similar until 180 days post-infection, with highly elevated IFN-gamma, IL-12 and IL-10 by d7 and decreasing thereafter. While both strains induced pneumonia and hepatitis in acute infection (d14), chronic infection (d56) was characterized, in addition to hepatitis, by severe meningoencephalitis, associated with numerous brain cysts. Thus, the BGD1 strain of T. gondii has type II genotypic and immunologic characteristics, but unlike other type II strains of human origin, induces severe encephalitis, making it an alternative to the sheep-derived ME49 strain for experimental models of infection.     

Competent dendritic cells derived from CD34+ progenitors express CMRF-44 antigen early in the differentiation pathway

Differentiation of CD34(+) haematopoietic stem cells into functional dendritic cells (DC) was investigated using the mAb CMRF-44 and other mAb against DC-associated markers. GM-CSF mobilized peripheral blood stem cells were obtained from healthy donors by leukapheresis. CD34(+) cells were purified using CD34(+)-positive selection,and subsequent immunomagnetic depletion of CD14 and CD2 cells. CD34(+) cells were cultured in medium supplemented with one or more of GM-CSF,TNF-alpha, IL-4 or IL-6. CMRF-44 Ag expression was monitored by flow cytometry, and DC function by allogeneic MLR and tetanus toxoid(TT) presentation assays. CD34(+) cells quickly acquired the CMRF-44 Ag when cultured in the presence of TNF-alpha.By day 3, more than 50% of the cells were double-positive for CD34 and CMRF-44. CD34 expression was gradually lost, so that by day 9, the majority of the cells were CD34(-)/CMRF-44(+).GM-CSF and TNF-alpha also induced CD40 expression, and up-regulation of CD54 and MHC class II on CD34(+) cells; their expression was correlated to the CMRF-44 Ag. Day 3 CD34(+)/CMRF-44(+) cells,but not CD34(+)/CMRF-44(-) cells, become potent APC when cultured further with GM-CSF plus TNF-alpha. These CMRF-44(+) cells were potent inducers of Th1-type immune response in the primary allogeneic MLR and present TT to autologous CD4(+) T cells. TNF-alpha alone is sufficient to induce CMRF-44 expression on CD34(+) cells, but in combination with GM-CSF expands the CMRF-44(+) population. CMRF-44 expression correlates with DC function and may be a useful early marker for commitment of CD34(+) cells to the DC differentiation pathway.