Dictyostelium discoideum nucleoside diphosphate kinase C plays a negative regulatory role in phagocytosis, macropinocytosis and exocytosis

Nucleoside diphosphate kinases (NDPKs) are ubiquitous phosphotransfer enzymes responsible for producing most of the nucleoside triphosphates except for ATP. This role is important for the synthesis of nucleic acids and proteins and the metabolism of sugars and lipids. Apart from this housekeeping role NDPKs have been shown to have many regulatory functions in diverse cellular processes including proliferation and endocytosis. Although the protein has been shown to have a positive regulatory role in clathrin- and dynamin-mediated micropinocytosis, its roles in macropinocytosis and phagocytosis have not been studied. The additional non-housekeeping roles of NDPK are often independent of enzyme activity but dependent on the expression level of the protein. In this study we altered the expression level of NDPK in the model eukaryotic organism Dictyostelium discoideum through antisense inhibition and overexpression. We demonstrate that NDPK levels affect growth, endocytosis and exocytosis. In particular we find that Dictyostelium NDPK negatively regulates endocytosis in contrast to the positive regulatory role identified in higher eukaryotes. This can be explained by the differences in types of endocytosis that have been studied in the different systems - phagocytosis and macropinocytosis in Dictyostelium compared with micropinocytosis in mammalian cells. This is the first report of a role for NDPK in regulating macropinocytosis and phagocytosis, the former being the major fluid phase uptake mechanism for macrophages, dendritic cells and other (non dendritic) cells exposed to growth factors.     

Features of uremic neuropathy in long-term dialysis

In order to assess the influence of long term hemodialysis on progression of uraemic neuropathy (UN), 16 different electroneurographic (ENG) parameters on 158 dialysis patients were performed. The ENG parameters were compared in three groups of patients of different dialysis age. Group I: high dialysis age (HDA) comprising of, 31 patients being more than 10 years on dialysis; Group II: intermediate dialysis age (IDA) comprising of 53 patients between 5 and 10 years on HD and group III: low dialysis age (LDA) comprising of 74 patients being less than 5 years on dialysis. The influence of sex and age was also analyzed. All sixteen tested parameters were altered in uremic patients when compared to 140 healthy controls (p < 0.01). HDA pts compared to LDA pts and the older group versus the younger had 11 and 9 out of 16 ENG parameters significantly worsened, respectively (p < 0.01). The most profound and reproducible lesion was in prolongation of evoked potential of tibialis posterior and peroneus nerve (FWt, HWt, FWp). HDA, especially after 10 yrs and the age but not the sex is clearly associated with a further progression of UN. However, for unknown reasons, the progression of UN in dialyzed patients is not followed by a parallel worsening of clinical symptoms (p > 0.05).     

Nuclei of symbiotic arbuscular mycorrhizal fungi as revealed by in vivo two-photon microscopy

The present work reports the results obtained from in vivo studies on the distribution and behavior of nuclei of two arbuscular mycorrhizal (AM) fungi growing in symbiosis with tomato root organ cultures (AM monoxenic cultures). Upon staining with 4',6-diamidino-2-phenylindole and two-photon microscopy (2PM) observations, symbiotic thick runner hyphae appeared mostly opaque to 2PM and did not reveal nuclei within them; thin runner hyphae showed dimly stained nuclei along them, whereas nuclei were clearly visible within the branches of the so-called branched absorbing structures. When visible, nuclei appeared anchored laterally at regular intervals along the symbiotic AM extraradical hyphae. Other nuclei migrate through the hyphal central core; this migration occurs in pulses. Simultaneous observations on different areas of extraradical AM mycelium revealed the existence of lysed compartments along the fungal hyphae, containing nuclei remnants and/or chromatin masses. All these results give new insights in (i) the differential permeability of AM hyphae in the symbiotic versus the asymbiotic state; (ii) the behavior and distribution of nuclei along the symbiotic extraradical mycelium; (iii) the occurrence of ageing events within the AM fungal colony; and (iv) the existence of "healing" mechanisms aiming to restrict the damage induced by such ageing or lytic events. An AM fungal strategy for hyphal survival under adverse conditions is also suggested.     

The Glyoxylate Cycle in an Arbuscular Mycorrhizal Fungus. Carbon Flux and Gene Expression

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.     

Putative sites for nutrient uptake in arbuscular mycorrhizal fungi

Nutrition of the arbuscular mycorrhiza (AM) is addressed from a fungal point of view. Intraradical and extraradical structures proposed as preferential sites for nutrient acquisition in arbuscular mycorrhizal (AM) fungi are considered, and their main features compared. This comparison includes the formation and function of branched structures (either intra- or extraradical) as putative nutrient uptake sites with unique morphological and physiological features in the AM fungal colony. The morphology and functioning of these structures are further affected by intra- or extraradical environmental factors. A model is presented which portrays the intrinsic developmental and physiological duality of the AM fungus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Competition and substrate colonization strategies of three polyxenically grown arbuscular mycorrhizal fungi

Intra- and extraradical colonization competition and hyphal interactions among arbuscular mycorrhizal fungi (AMF) Glomus intraradices, Glomus proliferum and Gigaspora margarita were investigated in two in vitro experimental systems. AMF were polyxenically cultured with a Ri T-DNA transformed carrot root organ culture (ROC) in either big Petri plates containing three culture compartments and a common hyphal compartment (i.e. an independent host root for each AMF) or two by two in the culture compartment of regular bicompartmented Petri dishes (i.e. a common host root and a common hyphal compartment). Maps of the extraradical mycelial development of the three AMF were obtained. Two distinct substrate colonization strategies (Glomus-type and Gigaspora-type) were identified, reflecting intrinsic differences among AMF genera/families. Our data reveal a general lack of antagonism between the isolates when extraradical hyphae explore and exploit the substrate outside the root influence zone; however certain growth restrictions were imposed by Gi. margarita extraradical mycelium when developing near the host root and by G. proliferum intraradical hyphae. This work highlights once more the appropriateness of AM in vitro culture systems to perform in vivo studies on the biology of this symbiosis and opens new avenues to the formulation of in vitro AMF inoculants.     

A simple method for the calculation of microsatellite genotype distances irrespective of ploidy level

Abstract Microsatellites are powerful molecular markers, used commonly to estimate intraspecific genetic distances. With the exception of band sharing similarity index, available distance measures were developed specifically for diploid organisms and are unsuited for comparisons of polyploids. Here, we present a simple method for calculation of microsatellite genotype distances, which takes into account mutation processes and permits comparison of individuals with different ploidy levels. This method should provide a valuable tool for intraspecific analyses of polyploid organisms, which are widespread among plants and some animal taxa. An illustration is given using data from the planarian flatworm Schmidtea polychroa (Platyhelminthes).     

Treatment of invasive cervical cancer: Rijeka experience

The aim of this retrospective analysis was to evaluate the survival rate in 661 patients with cervical cancer regarding two time periods 1990-1996 and 1997-2003 and the specific stage related risk factors. The respective five-year survival was 71.7% and 80.0%. Analyzing the risk factors in the univariate and multivariate regression modalities ultimately only two parameters, the two time periods and FIGO staging were found to be independent prognostic factors. The observed total improvement in the survival rate of the second time period is followed by an increase in conservative surgery in stage TIA1, a reduction in the use of adjuvant radiotherapy among operable stages Tlbl, Tlb2 and T2A, while the treatment of locally advanced cervical cancer did not differ significantly.