Molecular Detection of Phytophthora cryptogea on Calendula officinalis and Gerbera jamesonii Artificially Inoculated with Zoospores

In this work, a protocol for zoospores production of Phytophthora cryptogea , an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10⁵, 5 × 10⁴, 5 × 10³, 5 × 10², 5 × 10¹ zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold ( Calendula officinalis ) and gerbera ( Gerbera jamesonii ) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴, 5 × 10³ P. cryptogea  zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10⁵, 5 × 10⁴ P. cryptogea  zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.     

Periphyton nitrogenase activity as an indicator of wetland eutrophication: spatial patterns and response to phosphorus dosing in a northern Everglades ecosystem

The use of periphyton nitrogenase activity (biological N₂ fixation) as an indicator of wetland P impact was assessed using patterns of nutrient content (C, N, P, Ca, Mg, K, Fe, and Mn) and acetylene reduction (AR) in floating cyanobacterial periphyton mat (metaphyton) communities of a P-enriched portion of the Florida Everglades, USA (Water Conservation Area-2A, WCA-2A). Spatial patterns of nutrients indicate the enrichment of floating mat periphyton N, P, Fe, and K, and the reduction of Mn and TN:TP in enriched marsh areas. In highly enriched areas, floating mat periphyton AR was approximately threefold greater than that in less enriched, interior marsh zones. Multiple regression models indicated AR dependence on P in eutrophic WCA-2A areas while the AR of more interior marsh periphyton mats was more closely related to tissue levels of Ca and Fe. Nitrogenase activity of floating mat periphyton from P-loaded mesocosms revealed a significant enhancement of N₂ fixation in samples receiving approximately 2–3 mg P m⁻² of cumulative P dosing or with biomass TP content of 100–300 mg kg⁻¹. At P contents above the optimum, mat periphyton AR was suppressed possibly as a result of changes in species composition or increased levels of NH₄⁺. After 3 years of dosing, consistently high AR occurred only at low rates of P enrichment (0.4–0.8 g P m⁻² yr⁻¹), and the patterns appeared to be seasonal. These findings agree with the hypothesis that P availability is a key determinant of nitrogenase activity in aquatic systems, and thus, may support the use of periphyton nitrogenase to indicate P impacts in P-limited systems. These results also demonstrate the potential existence of a P threshhold for biogeochemical alteration of periphyton mat function in the Everglades, and that cumulative loading of limiting nutrients (i.e., P), rather than instantaneous concentrations, should be considered when evaluating nutrient criteria.     

Assessment of the ecological status of European surface waters: a work in progress

Medicine Lake is a small (165 ha), relatively shallow (average 7.3 m), intermediate elevation (2,036 m) lake located within the summit caldera of Medicine Lake volcano, Siskiyou County, California, USA. Sediment cores and high-resolution bathymetric and seismic reflection data were collected from the lake during the fall of 1999 and 2000. Sediments were analyzed for diatoms, pollen, density, grain size (sand/mud ratio), total organic carbon (TOC), and micro-scale fabric analysis. Using both ¹⁴C (AMS) dating and tephrochronology, the basal sediments were estimated to have been deposited about 11,400 cal year BP, thus yielding an estimated average sedimentation rate of about 20.66 cm/1,000 year. The lowermost part of the core (11,400–10,300 cal year BP) contains the transition from glacial to interglacial conditions. From about 11,000–5,500 cal year BP, Medicine Lake consisted of two small, steep-sided lakes or one lake with two steep-sided basins connected by a shallow shelf. During this time, both the pollen (Abies/Artemisia ratio) and the diatom (Cyclotella/Navicula ratio) evidences indicate that the effective moisture increased, leading to a deeper lake. Over the past 5,500 years, the pollen record shows that effective moisture continued to increase, and the diatom record indicates fluctuations in the lake level. The change in the lake level pattern from one of the increasing depths prior to about 6,000 cal year BP to one of the variable depths may be related to changes in the morphology of the Medicine Lake caldera associated with the movement of magma and the eruption of the Medicine Lake Glass Flow about 5,120 cal year BP. These changes in basin morphology caused Medicine Lake to flood the shallow shelf which surrounds the deeper part of the lake. During this period, the Cyclotella/Navicula ratio and the percent abundance of Isoetes vary, suggesting that the level of the lake fluctuated, resulting in changes in the shelf area available for colonization by benthic diatoms and Isoetes. These fluctuations are not typical of the small number of low-elevation Holocene lake records in the region, and probably reflect the hydrologic conditions unique to Medicine Lake.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis in vivo

Melanotransferrin is a member of the transferrin family, which is comprised of serum transferrin, lactoferrin and ovotransferrin, and is highly expressed on melanoma cells compared to normal melanocytes. Since melanoma is an highly vascularized tumour that expresses melanotransferrin at high levels, we tested purified recombinant melanotransferrin for its capability to induce angiogenesis in the chick chorioallantoic membrane. Macroscopic and microscopic evaluation of the vascular density demonstrated that melanotransferrin exerts an angiogenic response quantitatively similar to that elicited by fibroblast growth factor-2. Overexpression of vascular endothelial growth factor-receptor-2 was observed in newly formed vessels, suggesting that the angiogenic activity of melanotransferrin may depend on activation of endogenous vascular endothelial growth factor. In addition, when antibodies against vascular endothelial growth factor were included in the assay, the angiogenic response was inhibited by 50%. In a Boyden chamber assay purified recombinant melanotransferrin induced chemotactic migration of vascular cells, which was decreased in the the presence of anti-vascular endothelial growth factor antibodies suggesting an involvement of vascular endothelial growth factor present in endothelial cells also in this assay. However, melanotransferrin was found not to directly bind to integrin alphavbeta3 or the vascular endothelial growth factor-receptor-2 as assessed in a BlAcore assay. A possible correlation between vascularization occurring during melanoma progression and the expression of melanotransferrin and vascular endothelial growth factor was established by immunolocalization of the two factors in sections of melanoma at different clinical steps of melanoma progression. These latter data strongly imply that melanotransferrin may participate in the vascularization of solid tumours and that inhibition of melanotransferrin could form the basis for intervention in tumours which use this pathway.     

Comparative phenotypic,molecular, and virulence characterization of Vibrio parahaemolyticus O3:K6 isolates

Historically, Vibrio parahaemolyticus infections have been characterized by sporadic cases caused by multiple, diverse serotypes. However, since 1996, V. parahaemolyticus serotype O3:K6 strains have been associated with several large-scale outbreaks of illness, suggesting the emergence of a "new" group of organisms with enhanced virulence. We have applied three different molecular subtyping techniques to identify an appropriate method for differentiating O3:K6 isolates from other serotypes. Pulsed-field gel electrophoresis (PFGE) following NotI digestion differentiated seven closely related subtypes among O3:K6 and related strains, which were distinct from PFGE patterns for non-O3:K6 isolates. Ribotyping and tdh sequencing were less discriminatory than PFGE, but further confirmed close genetic relationships among recent O3:K6 isolates. In vitro adherence and cytotoxicity studies with human epithelial cells showed that O3:K6 isolates exhibited statistically higher levels of adherence and cytotoxicity to host cells than non-O3:K6 isolates. Epithelial cell cytotoxicity patterns were determined with a lactate dehydrogenase release assay. At 3 h postinfection, high relative cytotoxicities (>50% maximum lactate dehydrogenase activity) were found among a greater proportion of recently isolated O3:K6 and closely related strains (75%) than among the non-O3:K6 isolates (23%). A statistically significant relationship between adherence and cytotoxicity suggests that the pathogenic potential of some isolates may be associated with increased adherence to epithelial cells. Our findings suggest that enhanced adherence and cytotoxicity may contribute to the apparent unique pathogenic potential of V. parahaemolyticus O3:K6 strains.     

Adult neural stem cells: plasticity and developmental potential.

Stem cells play an essential role during the processes of embryonic tissue formation and development and in the maintenance of tissue integrity and renewal throughout adulthood. The differentiation potential of stem cells in adult tissues has been thought to be limited to cell lineages present in the organ from which they derive, but there is evidence that somatic stem cells may display a broader differentiation repertoire. This has been documented for bone marrow stem cells (which can give rise to muscle, hepatic and brain cells) and for muscle precursors, which can turn into blood cells. The adult central nervous system (CNS) has long been considered incapable of cell renewal and structural remodeling. Recent findings indicate that, even in postnatal and adult mammals, neurogenesis does occur in different brain regions and that these regions actually contain adult stem cells. These cells can be expanded both in vivo and ex vivo by exposure to different combinations of growth factors and subsequently give rise to a differentiated progeny comprising the major cell types of the CNS. Almost paradoxically, adult neural stem cells display a multipotency much broader than expected, since they can differentiate into non-CNS mesodermal-derivatives, such as blood cells and skeletal muscle cells. We review the recent findings documenting this unforeseen plasticity and unexpected developmental potential of somatic stem cells in general and of neural stem cells in particular. To better introduce these concepts, some basic notions on the functional properties of adult neural stem cells will also be discussed, particularly focusing on the emerging role of the microenvironment in determining and maintaining their peculiar characteristics.     

Cytotoxic activity of (S)-goniothalamin and analogues against human cancer cells

(R)- and (S)-Goniothalamin (1) and analogues 2-9 were efficiently prepared in high overall yield and enantiomeric purity, and their cytotoxic activities were evaluated against eight human cancer cell lines. A structure-activity relationship study (SAR) allowed us to establish the relevant structural features for the cytotoxic activity of goniothalamin analogues. In addition, we have identified non-natural form of goniothalamin (S)-1 and analogue 5 as the highest and more selective cytotoxic compounds against kidney cancer cell growth (786-0) with IC50 = 4 and 5 nM, respectively, and compound 8 (IC50 = 4 nM) as the more potent against breast cancer cells with resistance phenotype for adryamycin (NCI.ADR).     

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.