Identification of three urease accessory proteins that are required for urease activation in Arabidopsis

Urease is a nickel-containing urea hydrolase involved in nitrogen recycling from ureide, purine, and arginine catabolism in plants. The process of urease activation by incorporation of nickel into the active site is a prime example of chaperone-mediated metal transfer to an enzyme. Four urease accessory proteins are required for activation in Klebsiella aerogenes. In plants urease accessory proteins have so far been only partially defined. Using reverse genetic tools we identified four genes that are necessary for urease activity in Arabidopsis (Arabidopsis thaliana; ecotypes Columbia and N?ssen). Plants bearing T-DNA or Ds element insertions in either the structural gene for urease or in any of the three putative urease accessory genes AtureD, AtureF, and AtureG lacked the corresponding mRNAs and were defective in urease activity. In contrast to wild-type plants, the mutant lines were not able to support growth with urea as the sole nitrogen source. To investigate whether the identified accessory proteins would be sufficient to support eukaryotic urease activation, the corresponding cDNAs were introduced into urease-negative Escherichia coli. In these bacteria, urease activity was observed only when all three plant accessory genes were coexpressed together with the plant urease gene. Remarkably, plant urease activation occurred as well in cell-free E. coli extracts, but only in extracts from cells that had expressed all three accessory proteins. The future molecular dissection of the plant urease activation process may therefore be performed in vitro, providing a powerful tool to further our understanding of the biochemistry of chaperone-mediated metal transfer processes in plants.     

Population state of Echinometra lucunter (Echinoida: Echinometridae) and its accompanying fauna on Caribbean rocky littoral from Colombia

In order to know the current condition of the Echinometra lucunter population in the Colombian Caribbean, ten zones of the most representative rocky-shore were selected for sampling between November 2002 and May 2003. In each zone, four transects of 10.25 m2 were located parallel to the coast, and measured with a 0.25 m2 quadrant under the tide level. Two subsamples of 0.01 m2 were chosen from each quadrant, in order to determine the sea urchin heights and its associated fauna. This species was found in nine of the selected zones where the rocky-shore was of sedimentary origin and was absent in Punta Gloria because of the igneous origin of the rock, that the sea urchins cannot bore. The greatest average densities were obtained in Zapsurro and Inca Inca with 69 and 65 ind/m2 respectively; these are high values for the Caribbean Sea, whereas Acandi and Punta Betin had the lowest because of continental water discharges. The most frequent test diameter was between 25 and 40 mm (smaller on the western zones). The density of E. lucunter and of its associated fauna (including the endemic Ophiothrix synoecina) in the Santa Marta area decreased in the last decade.     

Immune response in cervical dysplasia induced by human papillomavirus: the influence of human immunodeficiency virus-1 co-infection -- review

Human immunodeficiency virus (HIV-1) has become an important risk factor for human papillomavirus (HPV) infection and the development of HPV associated lesions in the female genital tract. HIV-1 may also increase the oncogenicity of high risk HPV types and the activation of low risk types. The Center for Disease Control and Prevention declared invasive cervical cancer an acquired immunodeficiency virus (AIDS) defining illness in HIV positive women. Furthermore, cervical cancer happens to be the second most common female cancer worldwide. The host's local immune response plays a critical factor in controlling these conditions, as well as in changes in the number of professional antigen-presenting cells, cytokine, and MHC molecules expression. Also, the production of cytokines may determine which arm of the immune response will be stimulated and may influence the magnitude of immune protection. Although there are many studies describing the inflammatory response in HPV infection, few data are available to demonstrate the influence of the HIV infection and several questions regarding the cervical immune response are still unknown. In this review we present a brief account of the current understanding of HIV/HPV co-infection, emphasizing cervical immune response.     

Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis

We have isolated and sequenced the genes encoding the heat shock proteins 60 (Hsp60) and 70 (Hsp70) of the salmon pathogen Piscirickettsia salmonis. The sequence analysis revealed the expected two open reading frames that encode proteins with calculated molecular weights of 60,060 and 70,400. The proteins exhibit a 70-80% homology with other known prokaryotic Hsp60 and Hsp70 sequences. The coding regions have been expressed in E. coli as thioredoxin fusion proteins. Both recombinant proteins were shown to elicit a humoral response when injected intraperitoneally in Atlantic salmon and also conferred protection to fish challenged with P. salmonis. The present data will facilitate further studies on the involvement of heat shock proteins in protective immunity of fish to infection by P. salmonis and their potential use in recombinants vaccines against this intracellular pathogen.     

Determination of cell concentration in a plant cell suspension using a fluorescence microplate reader

Microscopic counting of plant cells is a very tedious and time-consuming process and is therefore seldom used to evaluate plant cell number on a routine basis. This study describes a fast and simple method to evaluate cell concentration in a plant cell suspension using a fluorescence microplate reader. Eschscholtzia californica cells were fixed in a mix of methanol and acetic acid (3:1) and stained with a fluorescent DNA binding dye (Hoechst 33258). Readings were done in a fluorescence microplate reader at 360/465 nm. Specific binding of the dye to double-stranded DNA was significantly favored over unspecific binding when 1.0 M Tris buffer at pH 7.5 containing 1.0 M NaCl and 75 microg ml(-1) of Hoechst 33258 was used. Fluorescence readings must be done between 4 min and 12 min following the addition of the staining solution to the sample. The microplate counting method provides a convenient, rapid and sensitive procedure for determining the cell concentration in plant cell suspensions. The assay has a linear detection range from 0.2 x 10(6) cells to 10.0 x 10(6) cells per milliliter (actual concentration in the tested cell suspension). The time needed to perform the microplate counting was 10% of that needed for the microscopic enumeration. However, this microplate counting method can only be used on genetically stable cell lines and on asynchronous cell suspensions.     

Host refractoriness of the tobacco hornworm, Manduca sexta, to the braconid endoparasitoid Cotesia flavipes

Cotesia flavipes (Hymenoptera:Braconidae) is a gregarious endoparasitoid of several pyralid stemborer larvae of economic significance including the sugarcane borer, Diatraea saccharalis. In this study, the ability of this parasitoid to develop in a sphingid host, Manduca sexta, was tested. First, second, third, fourth, and even pharate fifth instar host tobacco hornworm larvae were readily parasitized by the female C. flavipes parasitoids but no wasp larvae hatched from the eggs in this refractory host. Instead, the parasitoid eggs were invariably encapsulated by the host's hemocytes and, ultimately, no parasitoids emerged from tobacco hornworm hosts. The first stages of encapsulation were evident at 2 h post-parasitization of the host M. sexta larvae, when the beginning stages of capsule formation were seen. The developmental fate of the host larvae with encapsulated parasitoids was variable. Most succumbed as abnormally small fifth instars or as post-wandering prepupal animals, while a few developed normally to the pupal stage. Dissection of all the larvae or pupae with encapsulated wasp eggs showed evidence of hemocytic encapsulation and melanization of the C. flavipes eggs. This report describes the association between C. flavipes and M. sexta, which appears to be an excellent model system for studying the physiological processes accompanying wasp egg encapsulation that result in death of the host as well as the parasitoid. Since the parasitoid egg never hatches, the system offers an excellent opportunity to identify and study the effects of parasitoid-injected polydnavirus and venom on host physiology.     

Spatially resolved characterization of biogenic manganese oxide production within a bacterial biofilm

Pseudomonas putida strain MnB1, a biofilm-forming bacterial culture, was used as a model for the study of bacterial Mn oxidation in freshwater and soil environments. The oxidation of aqueous Mn+2 [Mn+2(aq)] by P. putida was characterized by spatially and temporally resolving the oxidation state of Mn in the presence of a bacterial biofilm, using scanning transmission X-ray microscopy (STXM) combined with near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the Mn L2,3 absorption edges. Subsamples were collected from growth flasks containing 0.1 and 1 mM total Mn at 16, 24, 36, and 48 h after inoculation. Immediately after collection, the unprocessed hydrated subsamples were imaged at a 40-nm resolution. Manganese NEXAFS spectra were extracted from X-ray energy sequences of STXM images (stacks) and fit with linear combinations of well-characterized reference spectra to obtain quantitative relative abundances of Mn(II), Mn(III), and Mn(IV). Careful consideration was given to uncertainty in the normalization of the reference spectra, choice of reference compounds, and chemical changes due to radiation damage. The STXM results confirm that Mn+2(aq) was removed from solution by P. putida and was concentrated as Mn(III) and Mn(IV) immediately adjacent to the bacterial cells. The Mn precipitates were completely enveloped by bacterial biofilm material. The distribution of Mn oxidation states was spatially heterogeneous within and between the clusters of bacterial cells. Scanning transmission X-ray microscopy is a promising tool for advancing the study of hydrated interfaces between minerals and bacteria, particularly in cases where the structure of bacterial biofilms needs to be maintained.