Paenibacillus larvae larvae spores in honey samples from Uruguay: a nationwide survey

American foulbrood is a severe bacterial disease affecting larvae of the honeybee Apis mellifera and it is caused by Paenibacillus larvae larvae. The disease is present worldwide and cases have been reported in almost all the beekeeping regions of the five continents. During 2001 and 2002 we carried out a nationwide study to assess the presence and amount of P. l. larvae spores in honey samples from Uruguay, combining classic bacteriological, and molecular approaches. The distribution of P. l. larvae spores in honey of the whole country showed a clear pattern and may provide useful data for a control and prevention strategy of American foulbrood.     

Folding of small RNAs displaying the GNC base-pattern: implications for the self-organization of the genetic system

Theoretical arguments and statistical analyses of present-day coding sequences have long suggested the existence of primitive patterns in RNA sequences, which were thought to have been predominant at the time of the origin of the genetic code. The main propositions were centered around the base-patterns GNC and RNY, where R = A or G , Y = C or U and N = A, G, C or U. A theoretical model of the primitive process of translation explaining the origin of this type of pattern was recently published in the Journal of Theoretical Biology. On the basis of this previous analysis, and on physico-chemical evidence supporting the idea of the GNC base-pattern as the most primitive one, the present paper shows the results of folding simulations of small RNA strands displaying this pattern, which enabled us to specify the characteristics of the suggested primitive form of tRNA. This analysis is notably based on a conjecture of Eigen and Schuster of an early structural (or pattern) similarity between mRNA and tRNA, and, more specifically, of a "joint function of messenger and adaptor". Working with this conjecture, we show that the convergence of the primitive pool of RNAs toward a system containing a high proportion of sequences displaying the GNC base-pattern (according to the evolutionary model) is accompanied by a significant gain in stability of the translation process. In particular, it is demonstrated how the reading frame would be automatically discriminated without the presence of a start codon.     

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.     

Cytokine gene expression in Walker 256: a comparison of variants A (aggressive) and AR (regressive)

Two variants of this Walker 256 tumor have been previously reported as Walker 256 A and variant AR. The variant A has more aggressive property than variant AR and can induce systemic effects such as anorexia, sodium and water retention, followed by weight loss and death. The mechanisms involved in enhancing tumor regression and progression in this model are still incompletely understood. In the present study, serum and spleen mononuclear cells and tumor cells from animals inoculated with variants A and AR, were isolated to investigate the TGF-beta, IL-12, IFN-gamma and TNF-alpha and relationship with anemia, weight of animals, weight of spleen, volume of tumor and osmotic fragility compared with controls inoculated with Ringer Lactate. Results demonstrate that the group inoculated with variant A, compared to variant AR, shows high levels of TGF-beta gene expression in both tumor tissue and spleen cells, no expression of IFN-gamma and a progressive and higher levels of IL-12 in tumor tissue without inflammatory infiltrate visualized by optical microscopy. These results suggest that the aggressively of variant A is relate to cytokine modulation, facilitating the growth and escape of tumor cells. Furthermore, IL-12 seems to be constitutively expressed in both tumor lineage A and AR.     

Proteomic analysis of high-density lipoprotein

Plasma lipoproteins, such as high-density lipoprotein (HDL), can serve as carriers for a wide range of proteins that are involved in processes such as lipid metabolism, thrombosis, inflammation and atherosclerosis. The identification of HDL-associated proteins is essential with regards to understanding these processes at the molecular level. In this study, a combination of proteomic approaches including 1-DE and 2-DE MALDI-TOF, isotope-coded affinity tag and Western blot analysis were employed to identify proteins associated with human HDL. To minimize potential losses of HDL-associated proteins during isolation, a one-step ultracentrifugation technique was applied and the quality of purified HDL was confirmed by nephelometry, high-performance gel chromatography, and Western blot analysis. MS analysis revealed the presence of 56 HDL-associated proteins including all known apolipoproteins and lipid transport proteins. Furthermore, proteins involved in hemostasis and thrombosis, the immune and complement system were found. In addition, growth factors, receptors, hormone-associated proteins and many other proteins were found to be associated with HDL. Our approach thus resulted in the identification of a large number of proteins associated with HDL. The combination of proteomic technologies proved to be a powerful and comprehensive tool for the identification of proteins on HDL.     

Comparative proteomic analysis of cisplatin sensitive IGROV1 ovarian carcinoma cell line and its resistant counterpart IGROV1-R10

Ovarian cancer is one of the leading causes of mortality due to gynaecological cancer. Despite a good response to surgery and initial chemotherapy essentially based on cisplatin (cis-diamino-dichloro-platinum(II) (CDDP)) compounds, late tumour detection and frequent recurrences with chemoresistance acquisition are responsible for poor prognosis. Several mechanisms have been implicated in CDDP resistance but they are not sufficient to exhaustively explain this resistance emergence. We applied a proteomic approach based on 2-DE coupled with MS to identify proteins associated with the chemoresistance process. We first established a proteomic pattern of the CDDP sensitive ovarian cell line IGROV1 using MALDI-TOF-MS and PMF. We then compared this 2-D pattern with that of the CDDP-resistant counterpart IGROV1-R10. Among the 40 proteins identified, cytokeratins 8 and 18 and aldehyde dehydrogenase 1 were overexpressed in IGROV1-R10, whereas annexin IV was down-regulated. These observations have been confirmed by Western blotting. The characterization of such variations could lead to the development of new protein markers or to the establishment of new therapeutic strategies. Moreover, the identification of proteins involved in CDDP resistance in ovarian tumours would be useful in completing our understanding on this complex mechanism.     

Biologically inspired sensing: infrared spectroscopic analysis of cell responses to an inhalation health hazard

This work describes the development of a biologically based sensing technique to quantify chemical agents that pose inhalation health hazards. The approach utilizes cultured epithelial cells (A549 human type II pneumocytes) of the lung, exposed to potential toxins and monitored through the noninvasive means of infrared spectroscopy to quantify changes to cell physiology and function. Cell response to Streptolysin O, a cholesterol-binding cytolysin, is investigated here. Infrared spectra display changes in cell physiology indicative of membrane damage, altered proteins, and some nucleic acid damage. Methods to improve cell adhesion through modification of support surface properties are detailed. This spectroscopic approach not only provides a robust means to detect potential toxins but also provides information on modes of damage and mechanisms of cellular response.     

PHProteomicDB： A Module for Two-dimensional Gel Electrophoresis Database Creation on Personal Web Sites

PHProteomicDB is a PHP-written module to help researchers in proteomics to share two-dimenslonal gel electrophoresis data using personal web sites. No technical or PHP knowledge is necessary except a few basics about web site management. PHProteomicDB has a user-friendly administration interface to enter and update data. It creates web pages on the fly displaying gel characteristics, gel pictures, and numbered gel spots with their related identifications pointing to their reference pages in protein databanks. The module is freely available at http：//www.huvec.com/index.php3？rub=Download.     

New Insights in Trichoderma harzianum Antagonism of Fungal Plant Pathogens by Secreted Protein Analysis

Trichoderma harzianum ALL42 were capable of overgrowing and degrading Rhizoctonia solani and Macrophomina phaseolina mycelia, coiling around the hyphae with formation of apressoria and hook-like structures. Hyphae of T. harzianum ALL42 did not show any coiling around Fusarium sp. hyphae suggesting that mycoparasitism may be different among the plant pathogens. In this study, a secretome analysis was used to identify some extracellular proteins secreted by T. harzianum ALL42 after growth on cell wall of M. phaseolina, Fusarium sp., and R. solani. The secreted proteins were analyzed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry. A total of 60 T. harzianum ALL42 secreted proteins excised from the gel were analyzed from the three growth conditions. While seven cell wall-induced proteins were identified, more than 53 proteins spots remain unidentified, indicating that these proteins are either novel proteins or proteins that have not yet been sequenced. Endochitinase, β-glucosidase, α-mannosidase, acid phosphatase, α-1,3-glucanase, and proteases were identified in the gel and also detected in the supernatant of culture.