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101.
The studies on the inhibitory effect exerted by Cholera Toxin (CT) on cell growth and proliferation indicate a remarkable heterogeneity of cell response suggesting that the inhibition represents the final event of many different ways or mechanisms . After CT binding, cAMP accumulation may not occur (as in L1210 leukemia cells) or, when occurring (as in SR-4987 stromal cells), may not be coupled with the antiproliferative effect of CT. In WEHI-3B cells CT binds a Gal-GalNac-GM1b receptor and the anticlonogenic effect of CT seems correlated with cAMP accumulation.To demonstrate the central role of cAMP in WEHI-3B cells, starting from the sensitive cell strain we selected and established a clone of WEHI-3B resistant to CT. This revertant clone (WEHI-3B/CT/REV) is currently cultured in the absence of CT and in the proliferation assay shows a dramatic resistance (>46,000 than the parental cells). Stimulation of WEHI-3B/CT/REV cells by cholera toxin failed to enhance cAMP and the ganglioside-CT binding studied on Thin Layer Chromatography (TLC) blots showed that the resistant cells lost the spot correspondent to the migration of Gal-GalNac-GM1b ganglioside. Both the lines respond at the same level to the adenylate cyclase stimulation by forskolin and the incorporation of GM1a did not decrease the resistance of WEHI-3B/CT/REV. These data confirm that Gal-GalNac-GM1b is the most important functional receptor for CT in WEHI-3B cells able to transduce the signal by enhancing cAMP which in turn inhibits cell proliferation (probably by cAMP dependent protein kinase activation). Our study describes the first cell line resistant to CT originated from a susceptible parental strain and provides a new interesting cell model for studying the cAMP dependent mechanisms involved in cell growth regulation. 相似文献
102.
103.
Zanotto C Elli V Basavecchia V Brivio A Paganini M Pinna D Vicenzi E De Giuli Morghen C Radaelli A 《FEMS immunology and medical microbiology》2003,35(1):59-65
Two different prime-boost immunization protocols were tested in rabbits and their immune response was evaluated and compared with the final aim of defining a vaccine strategy that might be able to protect non-human primates from infection with the pathogenic simian/human immunodeficiency virus, SHIV(89.6P). The two regimens were based on three priming immunizations with either an expression plasmid plus a fowlpox (FP) recombinant vector or with two FP recombinant vectors, each one expressing either the SIV(mac239) gag/pol or the HIV-1env(89.6P) genes. In both protocols, priming immunizations were followed by two boosts with SHIV-mimicking virus-like particles (VLP). A complete SHIV-specific response was observed in all animals. Interestingly, the DNA vaccine was three to 10 times more efficient than the FP recombinant in inducing an anti-gag humoral response. Real-time PCR confirmed the memory effect on T-cell subsets secreting interleukin-4 and interferon-gamma, as a consequence of stimulation of both arms of the immune system. Although both protocols were almost equally effective in eliciting homologous neutralizing antibodies and highlighted the efficacy of VLP administration for boosting, protocol A seemed to be more effective in promoting a balanced T-cell memory immune response and appears more promising for vaccine purposes. 相似文献
104.
The absence of a rapid and inexpensive method for estimating macroinvertebrate individual biomass is a limit to the study of freshwater communities, given that traditional methods are time- and money-consuming. We propose an inexpensive method to quickly estimate individual biomass from video-recorded images of pond macroinvertebrates. We used a software that automatically measures several body dimensions (area, perimeter, minor and major axes) on each individual and linear regression to relate these body dimensions to dry weight. Area was found to be the best predictor of dry weight. The method allows individual measures of macroinvertebrates at a reasonable speed and accuracy, and may be useful for biomass size spectra or secondary production studies. 相似文献
105.
Characterization of the hcnABC Gene Cluster Encoding Hydrogen Cyanide Synthase and Anaerobic Regulation by ANR in the Strictly Aerobic Biocontrol Agent Pseudomonas fluorescens CHA0 总被引:1,自引:0,他引:1 下载免费PDF全文
Jacques Laville Caroline Blumer Christine Von Schroetter Valeria Gaia Genevive Dfago Christoph Keel Dieter Haas 《Journal of bacteriology》1998,180(12):3187-3196
The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the −40 sequence (TTGGC….ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT….ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot. 相似文献
106.
Francesca Catanzano Giuseppe Graziano Valeria Cafaro Giuseppe D’Alessio Alberto Di Donato Guido Barone 《International journal of biological macromolecules》1998,23(4):277-285
Four residues Pro19, Leu28, Cys31 and Cys32 proved to be the minimal structural requirements in determining the dimeric structure and the N-terminal segment swapping of bovine seminal ribonuclease, BS-RNase. We analyzed the content of secondary and tertiary structures in RNase A, P-RNase A, PL-RNase A, MCAM-PLCC-RNase A and MCAM-BS-RNase, performing near and far-UV CD spectra. It results that the five proteins have very similar native conformations. Thermal denaturation at pH 5.0 of the proteins, studied by means of CD measurements, proved reversible and well represented by the two-state ND transition model. Thermodynamic data are discussed in the light of the structural information available for RNase A and BS-RNase. 相似文献
107.
Vittoria Petruzzella Valeria Tiranti Patricio Fernandez Paola Ianna Rosalba Carrozzo Massimo Zeviani 《Genomics》1998,54(3):494
We have successfully applied a strategy based on the “cyberscreening” of the expressed sequence tags database using yeast protein sequences as “probes” to identify the human gene orthologs to BCS1, COX15, PET112, COX11, and SCO1, five yeast genes involved in the biogenesis of the mitochondrial respiratory chain complexes. In yeast, BCS1 is involved mainly in the assembly of complex III, while the other genes appear to control the structure/function of cytochrome-coxidase. Significant amino acid identity and similarity were demonstrated by comparison of the human with the corresponding yeast polypeptides. Sequence alignment revealed numerous colinear identical regions and the conservation of functional domains. Mitochondrial targeting of the human gene products, suggested by computer analysis of the protein sequences, was confirmed by anin vitroimport and protease-protection assay. These data strongly suggest that the human gene products share similar or identical functions with their yeast homologues. Genes controlling the structure/function of the respiratory chain complexes are attractive candidates for human mitochondrial disorders such as Leigh disease. However, both sequence analysis and functional complementation assays on an index patient do not support an etiological role for any of these genes. 相似文献
108.
Samuele Raccosta Mauro Manno Donatella Bulone Daniela Giacomazza Valeria Militello Vincenzo Martorana Pier Luigi San Biagio 《European biophysics journal : EBJ》2010,39(6):1007-1017
The formation of protein aggregates is important in many fields of life science and technology. The morphological and mechanical
properties of protein solutions depend upon the molecular conformation and thermodynamic and environmental conditions. Non-native
or unfolded proteins may be kinetically trapped into irreversible aggregates and undergo precipitation or gelation. Here,
we study the thermal aggregation of lysozyme in neutral solutions. We characterise the irreversible unfolding of lysozyme
by differential scanning calorimetry. The structural properties of aggregates and their mechanisms of formation with the eventual
gelation are studied at high temperature by spectroscopic, rheological and scattering techniques. The experiments show that
irreversible micron-sized aggregates are organised into larger clusters according to a classical mechanism of diffusion and
coagulation, which leads to a percolative transition at high concentrations. At a smaller length scale, optical and atomic
force microscopy images reveal the existence of compact aggregates, which are the origin of the aggregation irreversibility. 相似文献
109.
Giuseppe Battaglia Milena Cannella Barbara Riozzi Sara Orobello Marion L. Maat‐Schieman Eleonora Aronica Carla Letizia Busceti Andrea Ciarmiello Silvia Alberti Enrico Amico Jenny Sassone Simonetta Sipione Valeria Bruno Luigi Frati Ferdinando Nicoletti Ferdinando Squitieri 《Journal of cellular and molecular medicine》2011,15(3):555-571
A defective expression or activity of neurotrophic factors, such as brain‐ and glial‐derived neurotrophic factors, contributes to neuronal damage in Huntington’s disease (HD). Here, we focused on transforming growth factor‐β (TGF‐β1), a pleiotropic cytokine with an established role in mechanisms of neuroprotection. Asymptomatic HD patients showed a reduction in TGF‐β1 levels in the peripheral blood, which was related to trinucleotide mutation length and glucose hypometabolism in the caudate nucleus. Immunohistochemical analysis in post‐mortem brain tissues showed that TGF‐β1 was reduced in cortical neurons of asymptomatic and symptomatic HD patients. Both YAC128 and R6/2 HD mutant mice showed a reduced expression of TGF‐β1 in the cerebral cortex, localized in neurons, but not in astrocytes. We examined the pharmacological regulation of TGF‐β1 formation in asymptomatic R6/2 mice, where blood TGF‐β1 levels were also reduced. In these R6/2 mice, both the mGlu2/3 metabotropic glutamate receptor agonist, LY379268, and riluzole failed to increase TGF‐β1 formation in the cerebral cortex and corpus striatum, suggesting that a defect in the regulation of TGF‐β1 production is associated with HD. Accordingly, reduced TGF‐β1 mRNA and protein levels were found in cultured astrocytes transfected with mutated exon 1 of the human huntingtin gene, and in striatal knock‐in cell lines expressing full‐length huntingtin with an expanded glutamine repeat. Taken together, our data suggest that serum TGF‐β1 levels are potential biomarkers of HD development during the asymptomatic phase of the disease, and raise the possibility that strategies aimed at rescuing TGF‐β1 levels in the brain may influence the progression of HD. 相似文献
110.
Notomista E Pennacchio F Cafaro V Smaldone G Izzo V Troncone L Varcamonti M Di Donato A 《Microbial ecology》2011,61(3):582-594
Novosphingobium sp. PP1Y, isolated from a surface seawater sample collected from a closed bay in the harbour of Pozzuoli (Naples, Italy),
uses fuels as its sole carbon and energy source. Like some other Sphingomonads, this strain can grow as either planktonic
free cells or sessile-aggregated flocks. In addition, this strain was found to grow as biofilm on several types of solid and
liquid hydrophobic surfaces including polystyrene, polypropylene and diesel oil. Strain PP1Y is not able to grow on pure alkanes
or alkane mixtures but is able to grow on a surprisingly wide range of aromatic compounds including mono, bi, tri and tetracyclic
aromatic hydrocarbons and heterocyclic compounds. During growth on diesel oil, the organic layer is emulsified resulting in
the formation of small biofilm-coated drops, whereas during growth on aromatic hydrocarbons dissolved in paraffin the oil
layer is emulsified but the drops are coated only if the mixtures contain selected aromatic compounds, like pyrene, propylbenzene,
tetrahydronaphthalene and heterocyclic compounds. These peculiar characteristics suggest strain PP1Y has adapted to efficiently
grow at the water/fuel interface using the aromatic fraction of fuels as the sole carbon and energy source. 相似文献