Functional analysis of an arthritogenic synovial fibroblast

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellullar matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.     

Protein farnesyltransferase and protein prenylation in Plasmodium falciparum

Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.     

Experimental study on photodynamic diagnosis of cancer mediated by chemiluminescence probe

A novel method of photodynamic diagnosis of cancer mediated by chemiluminescence probe is presented. The mechanism for photodynamic therapy involves singlet oxygen ((1)O(2)) generated by energy transfer from photosensitizers. (1)O(2) can react with 3,7-dihydro-6-[4-[2-(N'-(5-fluoresceinyl)thioureido)ethoxy]phenyl]-2-methylimidazo[1,2-a]pyrazin-3-one sodium salt (FCLA), which is a Cypridina luciferin analog and a specific chemiluminescence probe for detecting (1)O(2) and superoxide (O(2)(-)). The reaction of FCLA and (1)O(2) can give emission with peak wavelength at about 532 nm. In the present study, FCLA was chosen as an optical reporter of (1)O(2) produced from the photosensitization reaction of hematoporphyrin derivative in model solution and in nude mice with transplanted mammary cancer. Photosensitized chemiluminescence from the reaction of FCLA with (1)O(2) was detected by a highly sensitive Intensified Charge-Coupled Device detector. The chemiluminescence was markedly inhibited by the addition of 10 mmol/l sodium azide (NaN(3)) to the model solution and minor effects were observed at the addition of 10 micromol/l superoxide dismutase, 20 mmol/l mannitol and 100 microg/ml catalase, respectively, thus indicating that (1)O(2) generation from photosensitization reaction mainly results in light emission. Experiments in vivo with tumor-bearing mice showed a clear chemiluminescence image of tumor. The study suggests that this novel method may be applicable to the diagnosis of superficial tumors.     

Preliminary experience with resistin assessment in common population

Resistin is a signal peptide produced by adipose tissue. Mice models have confirmed that resistin may play an important role in insulin resistance. Its function in the human organism has not been elucidated yet. Since in common population the resistin concentrations are not known (no validated commercial set is available), we performed resistin assessment using the ELISA method (with satisfying analytical characteristics) in a population of 123 non-obese probands without signs of insulin resistance and/or inflammation. Mean resistin values amounted to 14.3 ng/ml (reference limit of 7.3-21.3 ng/ml).     

Photosynthesis and water relations of almond tree cultivars grafted on two rootstocks

Five cultivars of Prunus amygdalus Batsch (Ferragnes, Ferrastar, Marcona, Garrigues, and Non Pareil) grafted on two different rootstocks (Garrigues and GF677), and two cultivars (Ferraduel and Casa Nova) grafted on GF677, were grown for three years under rainfed conditions in an orchard in northeast Portugal. Net photosynthetic rate (PN), leaf conductance for water vapour (gs), leaf water potential (Ψ), instantaneous water use efficiency (WUE), and internal CO2 concentration (Ci) were measured at three periods of the growing season: spring, summer (June or July) and late summer (September) over two years. Ferraduel, Ferrastar, and Marcona presented the best performance in the periods when environmental conditions were not very hard (May or September). Casa Nova and Non Pareil were well adapted to high air evaporative demand, preventing the increase of leaf temperature (T1). Ferrastar, although having a good performance in May and September, did well adapt to hard climatic conditions in June 1994. In the following year, although it presented the highest T1, the values were not limiting (30.6 ± 2.1 °C), and PN was only decreased from May to July. Marcona was highly dependent on T1, but prevented its increasing. Garrigues showed lower PN in most measurement periods. GF677 frequently induced the highest PN, WUE, and Ψ. PN was mainly dependent on T1, radiation, Ci, month, and year. WUE depended on the same factors. Ψ depended mainly on gs, air temperature, month, and year.     

An autoselection system in recombinant Kluyveromyces lactis enhances cloned gene stability and provides freedom in medium selection

An autoselection system for increasing plasmid stability in Kluyveromyces lactis, based on the blockage of the pyrimidine de novo and salvage pathways, was investigated. In a manner analogous to that used in Saccharomyces cerevisiae, a putative “fur1” mutation was selected in a uraA K. lactis strain using 5-fluorouracil and 5-fluorocytosine plates. Survival of the mutant required expression of a plasmid-borne URA3 gene regardless of the culture medium employed, verifying the efficacy of this autoselection system in K. lactis. The expression of heterologous invertase, encoded by the S. cerevisiae SUC2 gene, was studied during long-term sequential batch cultures (70 generations) in complex yeast/peptone/glucose medium. The fur1 mutant successfully retained the plasmid; invertase specific activity remained above 90% of the initial level. Furthermore, no mutation reversion was observed. In contrast, for the control non-fur1 strain, only 4% of the cells retained the plasmid after 70 generations, and invertase specific activity dropped to less than 10% of the initial level. Experiments comparing growth and activity in different media indicated the potential for improving productivity through medium enrichment using this autoselection system. Received: 1 April 1997 / Received revision: 16 August 1997 / Accepted: 11 September 1997     

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

Effects of Temperature, Salinity, and Medium Composition on Compatible Solute Accumulation by Thermococcus spp.

The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identified as β-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered compound, T. litoralis accumulated mannosylglycerate, aspartate, α-glutamate, di-myo-inositol-1,1′(3,3′)-phosphate, hydroxyproline, and trehalose. The hydroxyproline and β-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1′(3,3′)-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated α-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of α-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1′(3,3′)-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo-inositol-1,1′(3,3′)-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.