uPA binding increases UPAR localization to lipid rafts and modifies the receptor microdomain composition

UPAR is a GPI anchored protein, which is found in both lipid rafts and in more fluid regions of the plasma membrane. We have studied the role of the ligand uPA on uPAR localization and on the composition of the lipid membrane microdomains. We have analyzed the glycosphingolipid environment of uPAR in detergent resistant membrane (DRM) fractions prepared by cell lysis with 1% Triton X-100 and fractionated by sucrose gradient centrifugation obtained from HEK293-uPAR cells. The uPAR specific lipid membrane microdomain has been separated from the total DRM fraction by immunoprecipitation with an anti-uPAR specific antibody under conditions that preserve membrane integrity. We have also tested uPA-induced ERK phosphorylation in the presence of methyl-beta-cyclodextrin, which is known to disrupt lipid rafts by sequestering cholesterol from such domains. Our results show that uPAR is partially associated with DRM and this association is increased by ligands, is independent of the catalytic activity of uPA, and is required for intracellular signalling. In the absence of ligands, uPAR experiences a lipid environment very similar to that of total DRM, enriched in sphingomyelin and glycosphingolipids. However, after treatment of cells with uPA or ATF the lipid environment is strongly impoverished of neutral glycosphingolipids.     

Sequence uniqueness and sequence variability as modulating factors of human anti-HCV humoral immune response

We recently compared the HCV polyprotein to the human proteome in order to test whether amino acid sequences unique to the virus could represent immunodominant epitopic determinants of the human humoral immune response against HCV. We identified a relatively limited number of HCV fragments with no/low similarity to the human host that represented exclusive HCV motifs. In this study, the peptides corresponding to low/zero similarity sequences were synthesized and assayed with HCV-infected sera. With different patterns, the synthetic HCV peptides corresponding to low/zero similarity sequences were found to be immunoreactive. In particular, the HCV E1 (315-323) HRMAWDMMM, HCV E2/NS1 (547-555) NWFGCTWMN, and HCV NS5 (2638-2646) YDTRCFDST sequences were immunodominant in the HCV-infected cohort under study. These three peptides correspond to sequences that are endowed with low-similarity to the human proteome, are highly conserved among various HCV strains, and have, potentially, a scarce susceptibility to proteolytic attacks. These data may be of help in defining the multiple factors which concur in the modulation of the human immune response against HCV, eventually providing information for the design of effective anti-HCV vaccines.     

Growth Factors and Steroid Mediated Regulation of Cytoskeletal Protein Expression in Serum-Deprived Primary Astrocyte Cultures

In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them togheter. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressfull conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Cardiovascular evaluation,including resting and exercise electrocardiography,before participation in competitive sports: cross sectional study

Objective To evaluate the clinical usefulness of complete preparticipation cardiovascular screening in a large cohort of sports participants.Design Cross sectional study of data over a five year period.Setting Institute of Sports Medicine in Florence, Italy.Participants 30 065 (23 570 men) people seeking to obtain clinical eligibility for competitive sports.Main outcome measures Results of resting and exercise 12 lead electrocardiography.Results Resting 12 lead ECG patterns showed abnormalities in 1812 (6%) participants, with the most common abnormalities (>80%) concerning innocent ECG changes. Exercise ECG showed an abnormal pattern in 1459 (4.9%) participants. Exercise ECG showed cardiac anomalies in 1227 athletes with normal findings on resting ECG. At the end of screening, 196 (0.6%) participants were considered ineligible for competitive sports. Among the 159 participants who were disqualified at the end of the screening for cardiac reasons, a consistent proportion (n=126, 79.2%) had shown innocent or negative findings on resting 12 lead ECG but clear pathological alterations during the exercise test. After adjustment for possible confounders, logistic regression analysis showed that age >30 years was significantly associated with an increased risk of being disqualified for cardiac findings during exercise testing.Conclusions Among people seeking to take part in competitive sports, exercise ECG can identify those with cardiac abnormalities. Follow-up studies would show if disqualification of such people would reduce the incidence of CV events among athletes.     

Assessing the value of secondary forest for amphibians: <Emphasis Type="Italic">Eleutherodactylus</Emphasis> frogs in a gradient of forest alteration

Secondary forests constitute a growing portion of forested areas worldwide. They might have a substantial role for the conservation of biodiversity in tropical areas, but there is little information on their potential to support forest species and the recovery of faunal communities. We studied two forest frogs (Eleutherodactylus diastema and E. fitzingeri) in an area of Costa Rica composed of a mosaic of primary forest, young secondary forest and pasture, and we compared the density of calling males in areas with different forest alteration. Autoregressive models were used to compensate for potentially undesired effects of spatial autocorrelation and pseudoreplication. Both species were most abundant in riparian, primary forest. However, E. fitzingeri was also abundant in riparian secondary forests, and its density far from the river was similar in primary and secondary forest, suggesting that river proximity can influence the recovery of secondary forest for amphibians. Conversely, the density of E. diastema was similar in secondary forest and pasture, stressing interspecific differences for recovery rate. These frogs have a keystone role in nutrient cycling and food webs, and their prompt recovery might represent an important step for the functional recovery of forests. Nevertheless, the strong interspecific differences stress the complexity of these processes.     

Delayed luminescence of Prorocentrum minimum under controlled conditions

Delayed luminescence (DL), also termed delayed fluorescence or delayed light emission, is the phenomenon of long-lived light emission by plants and cyanobacteria after being illuminated with light and put into darkness. Culture growth of three Prorocentrum minimum strains was studied with DL measurements. DL decay kinetics was measured from 1–60 s after a pulse of white light. The strains used were from the Adriatic Sea (PmK), from Chesapeake Bay, USA (D5), and from the Baltic Sea (BAL), cultured at salinity of 32, 16, and 8 (practical salinity scale), respectively. The strains differed in cell size and chlorophyll a content (PmK > D5 > BAL), as well as in DL parameters. The DL results were compared to standard measurements of culture density and carbon content (calculated from biovolumes). DL decay curves had a specific peak, which changed with culture growth and showed more similarities between the strains PmK and D5. The DL intensity increased with cell density and carbon content in a two-stage process, corresponding to the lag and exponential phases of growth. DL intensity was best correlated with carbon content irrespective of strain and is proposed as an estimate of biomass and for differentiating between lag and exponential phases of growth.     

A study in UF-membrane reactor on activity and stability of nitrile hydratase from Microbacterium imperiale CBS 498-74 resting cells for propionamide production

The bioconversion of propionitrile to propionamide was catalysed by nitrile hydratase (NHase) using resting cells of Microbacterium imperiale CBS 498-74 (formerly, Brevibacterium imperiale). This microorganism, cultivated in a shake flask, at 28 °C, presented a specific NHase activity of 34.4 U mg_DCW⁻¹ (dry cell weight). The kinetic parameters, K_m and V_max, tested in 50 mM sodium phosphate buffer, pH 7.0, in the propionitrile bioconversion was evaluated in batch reactor at 10 °C and resulted 21.6 mM and 11.04 μmol min⁻¹ mg_DCW⁻¹, respectively. The measured apparent activation energy, 25.54 kJ mol⁻¹, indicated a partial control by mass transport, more likely through the cell wall.

UF-membrane reactors were used for kinetic characterisation of the NHase catalysed reaction. The time dependence of enzyme deactivation on reaction temperature (from 5 to 25 °C), on substrate concentrations (from 100 to 800 mM), and on resting cell loading (from 1.5 to 200 μg  ml⁻¹) indicated: lower diffusional control (E_a=37.73 kJ mol⁻¹); and NHase irreversible damage caused by high substrate concentration. Finally, it is noteworthy that in an integral reactor continuously operating for 30 h, at 10 °C, 100% conversion of propionitrile (200 mM) was attained using 200 μg  ml⁻¹ of resting cells, with a maximum volumetric productivity of 0.5 g l⁻¹ h⁻¹.     

Differential response of linear and angular psoralens in PUVA-induced apoptosis in HL-60 cells.

Treatment of cells with UVA radiation in combination with linear psoralens induces a cell-cycle block and subsequent apoptosis, whereas angular derivatives produce apoptosis without affecting the cellular cycle.     

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.