Airborne pollen in Padua (NE-Italy): A comparison between two pollen samplers

During recent years a gradual decrease inallergenic airborne pollen concentration hasbeen observed in the monitoring station ofPadua (Italy). Because technical checks of thesampler were not able to explain this trend,the results obtained from two twinpollen-samplers (Lanzoni VPPS 2000), placed twometres apart, were compared.An eight-week sampling was carried out duringthe year 2000 from July to September.Subsequent analysis revealed no statisticallysignificant difference between the dataobtained with the two instruments. On the otherhand, both samplers captured high levels offungal spores. We conclude that the observednegative trend in pollen count is real and notrelated to technical biases.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Increased kynurenine-to-tryptophan ratio in the serum of patients infected with SARS-CoV2: An observational cohort study.

Immune dysregulation is a hallmark of patients infected by SARS-CoV2 and the balance between immune reactivity and tolerance is a key determinant of all stages of infection, including the excessive inflammatory state causing the acute respiratory distress syndrome. The kynurenine pathway (KP) of tryptophan (Trp) metabolism is activated by pro-inflammatory cytokines and drives mechanisms of immune tolerance. We examined the state of activation of the KP by measuring the Kyn:Trp ratio in the serum of healthy subjects (n = 239), and SARS-CoV2-negative (n = 305) and -positive patients (n = 89). Patients were recruited at the Emergency Room of St. Andrea Hospital (Rome, Italy). Kyn and Trp serum levels were assessed by HPLC/MS-MS. Compared to healthy controls, both SARS-CoV2-negative and -positive patients showed an increase in the Kyn:Trp ratio. The increase was larger in SARS-CoV2-positive patients, with a significant difference between SARS-CoV2-positive and -negative patients. In addition, the increase was more prominent in males, and positively correlated with age and severity of SARS-CoV2 infection, categorized as follows: 1 = no need for intensive care unit (ICU); 2 ≤ 3 weeks spent in ICU; 3 ≥ 3 weeks spent in ICU; and 4 = death. The highest Kyn:Trp values were found in SARS-CoV2-positive patients with severe lymphopenia. These findings suggest that the Kyn:Trp ratio reflects the level of inflammation associated with SARS-CoV2 infection, and, therefore, might represent a valuable biomarker for therapeutic intervention.     

The Helicobacter pylori VacA cytotoxin activates RBL-2H3 cells by inducing cytosolic calcium oscillations

Helicobacter pylori causes an acute inflammatory response followed by chronic infection of the human gastric mucosa. Identification of the bacterial molecules endowed with a pro-inflammatory activity is essential to a molecular understanding of the pathogenesis of H. pylori associated diseases. The vacuolating cytotoxin A (VacA) induces mast cells to release pro-inflammatory cytokines. Here, we show that VacA activates the mast cell line RBL-2H3 by rapidly inducing an oscillation of the level of cytosolic calcium with exocytosis of secretory granules. Cytosolic calcium derives mainly from intracellular stores. VacA also stimulates a calcium-dependent production of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-alpha). These observations indicate that VacA may act as a pro-inflammatory factor of H. pylori at very early stages of the innate immune response.     

Structural Integrity of the Contralesional Hemisphere Predicts Cognitive Impairment in Ischemic Stroke at Three Months

After stroke, white matter integrity can be affected both locally and distally to the primary lesion location. It has been shown that tract disruption in mirror’s regions of the contralateral hemisphere is associated with degree of functional impairment. Fourteen patients suffering right hemispheric focal stroke (S) and eighteen healthy controls (HC) underwent Diffusion Weighted Imaging (DWI) and neuropsychological assessment. The stroke patient group was divided into poor (SP; n = 8) and good (SG; n = 6) cognitive recovery groups according to their cognitive improvement from the acute phase (72 hours after stroke) to the subacute phase (3 months post-stroke). Whole-brain DWI data analysis was performed by computing Diffusion Tensor Imaging (DTI) followed by Tract Based Spatial Statistics (TBSS). Assessment of effects was obtained computing the correlation of the projections on TBSS skeleton of Fractional Anisotropy (FA) and Radial Diffusivity (RD) with cognitive test results. Significant decrease of FA was found only in right brain anatomical areas for the S group when compared to the HC group. Analyzed separately, stroke patients with poor cognitive recovery showed additional significant FA decrease in several left hemisphere regions; whereas SG patients showed significant decrease only in the left genu of corpus callosum when compared to the HC. For the SG group, whole brain analysis revealed significant correlation between the performance in the Semantic Fluency test and the FA in the right hemisphere as well as between the performance in the Grooved Pegboard Test (GPT) and theTrail Making Test-part A and the FA in the left hemisphere. For the SP group, correlation analysis revealed significant correlation between the performance in the GPT and the FA in the right hemisphere.     

Protein synthesis meets ABC ATPases: new roles for Rli1/ABCE1

The essential NTPase Rli1/ABCE1 has been implicated in translation initiation, ribosome biogenesis, and human immunodeficiency virus capsid assembly. Two recent papers by the Krebber and Pestova groups —the former published in this issue of EMBO reports— suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes.EMBO Rep (2010) 11: 3 214–219. doi:10.1038/embor.2009.272The essential, conserved NTPase Rli1/ABCE1—a member of the ABC (ATP-binding cassette) superfamily of ATPases—has been implicated in translation initiation, ribosome biogenesis and human immunodeficiency virus capsid assembly. Two recent papers by the Krebber and Pestova groups—the former published in this issue of EMBO reports—suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes (Khoshnevis et al, 2010; Pisarev et al, 2010).Two recent papers […] suggest new important roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotesProtein synthesis is divided into four phases—initiation, elongation, termination and ribosome recycling—which are catalysed by several translation factors. The fundamental reactions of protein synthesis, such as mRNA decoding, peptide bond formation and tRNA translocation, follow the same basic principles in prokaryotes and eukaryotes. However, some steps are quite different and require a larger set of factors in eukaryotes. The best-studied example of eukaryotic complexity is the initiation of protein synthesis. In prokaryotes, initiation is catalysed by only three factors—IF1, IF2 and IF3—whereas in mammals it requires at least 13. Two recent papers shed new light on termination and ribosome recycling in the yeast and mammalian systems, suggesting that these two steps are also different in eukaryotes and prokaryotes (Khoshnevis et al, 2010; Pisarev et al, 2010).…new [research] on termination and ribosome recycling in the yeast and mammalian systems [suggests] that these two steps are also different in eukaryotes and prokaryotesIn prokaryotes, translation termination is promoted by three release factors: RF1, RF2 and RF3. RF1 and RF2 recognize the three stop codons and catalyse the hydrolysis of the peptidyl-tRNA. RF3, a GTP-binding protein that is not essential in bacteria, does not participate in peptide release but, at the expense of GTP hydrolysis, promotes the dissociation of RF1 and RF2, thereby accelerating their turnover (Kisselev et al, 2003). To free the ribosome for initiation on another mRNA (a process known as recycling), the post-termination ribosome is disassembled in a step that requires ribosome recycling factor (RRF) and one of the elongation factors, the GTPase EF-G. Together, these factors promote the dissociation of the post-termination complex into subunits. The subsequent dissociation of tRNA and mRNA from the small ribosomal subunit is promoted by initiation factors, in particular IF3 (Peske et al, 2005).In eukaryotes, translation termination is mediated by only two factors: eRF1 recognizes all three termination codons and triggers the hydrolysis of peptidyl-tRNA, whereas eRF3 accelerates the process in a GTP-dependent manner (Fig 1; Alkalaeva et al, 2006). Unlike prokaryotic RF1 or RF2—which have no measurable affinity for RF3—eRF1 binds tightly to eRF3, and it is probably the complex of the two proteins that enters the ribosome. The mechanism of guanine nucleotide exchange on eRF3 is also different from that on prokaryotic RF3, suggesting that termination in eukaryotes and prokaryotes differs in almost every detail except, probably, the mechanism of peptidyl-tRNA hydrolysis itself. Nevertheless, the identification of an additional factor that facilitates termination was unexpected. In this issue of EMBO reports, Khoshnevis and colleagues use the power of yeast genetics to show that a protein named Rli1 (RNase L inhibitor 1) interacts physically with the termination factors eRF1 (known as Sup45 in yeast) and, to a lesser extent, eRF3 (Sup35; Khoshnevis et al, 2010). The downregulation of Rli1 expression increases stop codon read-through in a dual reporter system, indicating a lower efficiency of termination. Conversely, upregulation of Rli1 partly suppresses the increased read-through caused by certain mutations of eRF1. Although the mechanism by which Rli1 affects translation termination is not understood, the results of the Krebber lab provide strong evidence that Rli1 mediates the function of eRF1 and eRF3 in vivo (Fig 1).…the identification [in eukaryotes] of an additional factor that facilitates termination was unexpectedOpen in a separate windowFigure 1New roles of Rli1/ABCE1 in translation termination and ribosome recycling in eukaryotes. During termination, translating ribosomes contain peptidyl-tRNA (peptide is shown in dark blue and tRNA in dark red) in the P site and expose a stop codon in the A site. The stop codon is recognized by termination factor eRF1, which enters the ribosome together with eRF3-GTP. After GTP hydrolysis, catalysed by eRF3, the peptide is released from the peptidyl-tRNA with the help of eRF1. The point at which Rli1/ABCE1 binds to the ribosome is unknown, but the order shown is consistent with the effect of the factor on both termination and recycling. After NTP hydrolysis by Rli1/ABCE1, the 60S subunit and factors dissociate from the 40S subunit. Finally, tRNA and mRNA are released from the 40S subunit with the help of initiation factors (not shown). ABCE1, ATP-binding cassette, sub-family E member 1; eRF, eukaryotic release factor; Rli1, RNase L inhibitor 1.Surprisingly, modulating the efficiency of termination seems not to be the only function of Rli1 in translation. In a parallel study, Pestova and co-workers show that in higher eukaryotes, the homologue of Rli1—ABCE1—strongly enhances ribosome recycling (Pisarev et al, 2010). Eukaryotes lack a homologue of bacterial RRF and thus have to use other factors to disassemble the post-termination ribosome. Ribosome recycling can be brought about to some extent by eIF3, eIF1 and eIF1A (Pisarev et al, 2007), which is reminiscent of the IF3/IF1-mediated slow ribosome recycling that seems to occur in some conditions in bacterial systems. In eukaryotes, the initiation-factor-driven recycling operates only in a narrow range of low Mg²⁺ concentrations, probably because the affinity of the subunits to one another increases steeply with Mg²⁺ (Pisarev et al, 2010). By contrast, ABCE1 seems to catalyse efficient subunit dissociation in various conditions. To bind to the ribosome, ABCE1 requires the presence of eRF1, which is thought to induce a conformational change of the ribosome that unmasks the binding site for ABCE1. Subunit dissociation requires NTP (ATP, GTP, CTP or UTP) hydrolysis by ABCE1 (Fig 1). Subsequently, the dissociation of mRNA and tRNA from the small ribosomal subunit is promoted by initiation factors, which also inhibit the spontaneous reassociation of the subunits. Thus, the sequence of events during ribosome recycling in the eukaryotic system is remarkably similar to that in prokaryotes, and ABCE1 and eRF1 (possibly together with eRF3) seem to act as genuine ribosome recycling factors, similar to bacterial RRF/EF-G, despite the lack of any similarity in sequence or structure.Rli1/ABCE1 is a member of the ABC ATPases and comprises four structural domains (Karcher et al, 2008). Two nucleotide-binding domains (1 and 2) are connected by a hinge and arranged in a head-to-tail orientation. In contrast to other ABC enzymes, ABCE1 has an amino-terminal iron–sulphur (Fe–S) cluster domain, which is located in close proximity to, and presumably interacts with the nucleotide-binding loop of domain 1. Thus, there is a potential link between Fe–S domain function and NTP-induced conformational control of the ABC tandem cassette. Interestingly, although Khosnevis and colleagues map the eRF1 binding site on the second, carboxy-terminal ATPase domain, the Fe–S cluster is required for the function of Rli1/ABCE1 in termination and recycling (Khoshnevis et al, 2010). One might speculate that NTP hydrolysis is coupled to splitting the ribosome into subunits, in analogy to the prokaryotic recycling factors RRF/EF-G that couple the free energy of GTP hydrolysis and phosphate release into subunit dissociation (Savelsbergh et al, 2009). Kinetic experiments measuring single-round rates of subunit dissociation and NTP hydrolysis would be required to establish the existence and nature of such coupling.Another intriguing question is the role of the Fe–S cluster in Rli1/ABCE1. Fe–S protein biogenesis is the only known function of mitochondria that is indispensable for the viability of yeast cells (Lill, 2009). As yeast mitochondria do not contain essential Fe–S proteins, the essential character of the mitochondrial Fe–S protein assembly machinery could be attributed to its role in the maturation of extra-mitochondrial Fe–S proteins, such as Rli1/ABCE1, which is essential in all organisms tested.Another interesting finding by the Krebber group is that Rli1 can bind to Hcr1 (known as eIF3j in higher eukaryotes; Khoshnevis et al, 2010). Hcr1/eIF3j is an RNA-binding subunit of initiation factor eIF3, which is involved in initiation and required for Rli1/ABCE1-independent ribosome recycling. The fact that Rli1/ABCE1 binds to both eRF1 and Hcr1/eIF3j might indicate a functional or regulatory link between the termination, recycling and initiation machineries eukaryotes. It is unclear why eukaryotes require termination and recycling machinery that is so different from that of prokaryotes. One possibility is that Rli1/ABCE1, in contrast to its prokaryotic counterparts, not only acts in termination and recycling but also provides a platform for the recruitment of initiation factors to the ribosome, thereby acting as an additional checkpoint for translational control. Thus, the results of the Krebber and Pestova labs open a new, exciting avenue of research on eukaryotic protein synthesis.     

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.     

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.     

Neutrophil beta2 integrin inhibition by enhanced interactions of vasodilator-stimulated phosphoprotein with S-nitrosylated actin

Production of reactive species in neutrophils exposed to hyperoxia causes S-nitrosylation of β-actin, which increases formation of short actin filaments, leading to alterations in the cytoskeletal network that inhibit β(2) integrin-dependent adherence (Thom, S. R., Bhopale, V. M., Mancini, D. J., and Milovanova, T. N. (2008) J. Biol. Chem. 283, 10822-10834). In this study, we found that vasodilator-stimulated protein (VASP) exhibits high affinity for S-nitrosylated short filamentous actin, which increases actin polymerization. VASP bundles Rac1, Rac2, cyclic AMP-dependent, and cyclic GMP-dependent protein kinases in close proximity to short actin filaments, and subsequent Rac activation increases actin free barbed end formation. Using specific chemical inhibitors or reducing cell concentrations of any of these proteins with small inhibitory RNA abrogates enhanced free barbed end formation, increased actin polymerization, and β(2) integrin inhibition by hyperoxia. Alternatively, incubating neutrophils with formylmethionylleucylphenylalanine or 8-bromo-cyclic GMP activates either cyclic AMP-dependent or cyclic GMP-dependent protein kinase, respectively, outside of the short F-actin pool and phosphorylates VASP on serine 153. Phosphorylated VASP abrogates the augmented polymerization normally observed with S-nitrosylated actin, VASP binding to actin, elevated Rac activity, and elevated formation of actin free barbed ends, thus restoring normal β(2) integrin function.