Wide-spectrum profile of inflammatory mediators in the plasma and scales of patients with psoriatic disease

Psoriasis is a chronic recurrent inflammatory disorder of the skin. Clinical subtypes include psoriasis vulgaris (PV), psoriatic arthropathy, and erythrodermic psoriasis. Aim of this study was to analyse relevant inflammatory mediators in the plasma of patients with distinct subtypes of active psoriasis, and in the scales of mild-to-moderate PV patients, and correlation to disease severity. Compared to healthy controls (n = 10), patients affected by very severe forms of psoriasis (n = 30) were characterized by increased plasma levels of IL-4, IL-6, MCP-1, VEGF and in particular PDGFbb. Each group with severe psoriasis had distinct characteristic features of plasma cytokine profile. Mild-to-moderate PV patients (n = 35) showed higher levels of IL-4, IL-6, IL-10, and IL-13 when compared to healthy controls. No correlation was found between PV severity assessed by PASI (Psoriasis Area and Severity Index) and levels of these mediators. By contrast, disease severity correlated to scale levels of IP-10. For the first time, we found exaggerated circulating levels of the pro-angiogenic PDGFbb and VEGF in severe psoriasis. Evidence that the severity of skin symptoms correlated exclusively with scale levels of IP-10, but not with any up-regulated inflammatory mediator in plasma, suggests that distinct skin-independent processes contribute to the circulating cytokine profile in psoriasis.     

Methodologies for social life cycle assessment

Goal, Scope and Background  

In recent years several different approaches towards Social Life Cycle Assessment (SLCA) have been developed. The purpose of this review is to compare these approaches in order to highlight methodological differences and general shortcomings. SLCA has several similarities with other social assessment tools, although, in order to limit the expanse of the review, only claims to address social impacts from an LCA-like framework are considered.     

SHOC1, an XPF endonuclease-related protein, is essential for the formation of class I meiotic crossovers

Crossovers (COs) are essential for the completion of meiosis in most species and lead to new allelic combinations in gametes. Two pathways of meiotic crossover formation have been distinguished. Class I COs, which are the major class of CO in budding yeast, mammals, Caenorhabditis elegans, and Arabidopsis, depend on a group of proteins called ZMM and rely on specific DNA structure intermediates that are processed to form COs. We identified a novel gene, SHOC1, involved in meiosis in Arabidopsis. Shoc1 mutants showed a striking reduction in the number of COs produced, a similar phenotype to the previously described Arabidopsis zmm mutants. The early steps of recombination, revealed by DMC1 foci, and completion of synapsis are not affected in shoc1 mutants. Double mutant analysis showed that SHOC1 acts in the same pathway as AtMSH5, a conserved member of the ZMM group. SHOC1 is thus a novel gene required for class I CO formation in Arabidopsis. Sequence similarity studies detected putative SHOC1 homologs in a large range of eukaryotes including human. SHOC1 appears to be related to the XPF endonuclease protein family, which suggests that it is directly involved in the maturation of DNA intermediates that lead to COs.     

A critical role for sphingosine kinase in anaphylatoxin-induced neutropenia, peritonitis, and cytokine production in vivo

The aim of our study was to investigate the roles played by sphingosine kinase (SPHK) in the anaphylatoxin C5a-triggered responses in vivo. Our data show that i.v. administration of C5a triggers a rapid neutropenic response, but pretreating mice with the SPHK inhibitor, N,N-dimethylsphingosine (DMS), 10 min before the C5a i.v. administration substantially inhibited the C5a-triggered neutropenia. Similarly the i.v. administration of C5a caused a rapid increase in the serum levels of TNF-alpha and IL-6, and this increase in cytokine levels was blocked by DMS. We then induced acute peritonitis with C5a. The C5a i.p. injection triggered a fast recruitment of neutrophils, later followed by monocytes, into the peritoneal cavity. Vascular permeability was also observed: when we i.v. injected Evans blue before C5a i.p. injection, we could observe a continued influx of the dye into the peritoneum. In mice pretreated with DMS, there was a significant reduction on the C5a-triggered neutrophil and monocyte infiltration, as well as a marked reduction on the Evans blue influx. Our data also show that the i.p. administration of C5a caused a rapid increase in TNF-alpha and IL-6 levels in the peritoneal cavity, and this increase in cytokine levels was substantially inhibited in mice pretreated with the SPHK inhibitor. Taken together, these observations suggest a potential role for SPHK in the C5a-triggered inflammatory responses in vivo.     

The Epstein-Barr virus protein, latent membrane protein 2A, co-opts tyrosine kinases used by the T cell receptor

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several human malignancies. The EBV protein latent membrane protein 2A (LMP2A) promotes viral latency in memory B cells by interfering with B cell receptor signaling and provides a survival signal for mature B cells that have lost expression of surface immunoglobulin. The latter function has suggested that LMP2A may enhance the survival of EBV-positive tumors. EBV is associated with several T cell malignancies and, since LMP2A has been detected in several of these disorders, we examined the ability of LMP2A to transmit signals and interfere with T cell receptor signaling in T cells. We show that LMP2A is tyrosine-phosphorylated in Jurkat TAg T cells, which requires expression of the Src family tyrosine kinases, Lck and Fyn. Lck and Fyn are recruited to the tyrosine-phosphorylated Tyr112 site in LMP2A, whereas phosphorylation of an ITAM motif in LMP2A creates a binding site for the ZAP-70/Syk tyrosine kinases. LMP2A also associates through its two PPPPY motifs with AIP4, a NEDD4 family E3 ubiquitin ligase; this interaction results in ubiquitylation of LMP2A and serves to regulate the stability of LMP2A and LMP2A-kinase complexes. Furthermore, stable expression of LMP2A in Jurkat T cells down-regulated T cell receptor levels and attenuated T cell receptor signaling. Thus, through recruiting tyrosine kinases involved in T cell receptor activation, LMP2A may provide a survival signal for EBV-positive T cell tumors, whereas LMP2A-associated NEDD4 E3 ligases probably titer the strength of this signal.     

Heat shock prevents alpha-synuclein-induced apoptosis in a yeast model of Parkinson's disease

We show that human wild-type alpha synuclein (WT alpha-syn), and the inherited mutants A53T or A30P, when expressed in the yeast Saccharomyces cerevisiae triggers events that are diagnostic of apoptosis: loss of membrane asymmetry due to the externalization of phosphatidylserine, accumulation of reactive oxygen species (ROS), and the release of cytochrome c from mitochondria. A brief heat shock was strikingly protective in that alpha-syn-expressing cells receiving a heat shock exhibited none of these apoptotic markers. Because the heat shock did not decrease the expression level of alpha-syn, a protective protein or proteins, induced by the heat shock, must be responsible for inhibition of alpha-syn-induced apoptosis. Using ROS accumulation as a marker of apoptosis, the role of various genes and various drugs in controlling alpha-syn-induced apoptosis was investigated. Treatment with geldanamycin or glutathione, overexpression of Ssa3 (Hsp70), or deletion of the yeast metacaspase gene YCA1 abolishes the ability of alpha-syn to induce ROS accumulation. Deletion of YCA1 also promotes vigorous growth of alpha-syn-expressing cells compared to cells that contain a functional copy of YCA1. These findings indicate that alpha-syn-induced ROS generation is mediated by the caspase, according to alpha-syn-->caspase-->ROS-->apoptosis. It is shown by co-immunoprecipitation that Ssa3 binds to alpha-syn in a nucleotide-dependent manner. Thus, we propose that Hsp70 chaperones inhibit this sequence of events by binding and sequestering alpha-syn.     

Mice deficient in complement receptors 1 and 2 lack a tissue injury-inducing subset of the natural antibody repertoire

Intestinal ischemia-reperfusion (IR) injury is initiated when natural Abs recognize neoantigens that are revealed on ischemic cells. Cr2(-/-) mice, deficient in complement receptors (CR)1 and CR2, demonstrate defects in T-dependent B-2 B cell responses to foreign Ags and have also been suggested to manifest abnormalities of the B-1 subset of B lymphocytes. To determine whether these CRs might play a role in the generation of the natural Abs that initiate intestinal IR injury, we performed experiments in Cr2(-/-) and control Cr2(+/+) mice. We found that Cr2(-/-) mice did not demonstrate severe intestinal injury that was readily observed in control Cr2(+/+) mice following IR, despite having identical serum levels of IgM and IgG. Pretreatment of Cr2(-/-) mice before the ischemic phase with IgM and IgG purified from the serum of wild-type C57BL/6 mice reconstituted all key features of IR injury, demonstrating that the defect involves the failure to develop this subset of natural Abs. Pretreatment with IgM and IgG individually demonstrates that each contributes to unique features of IR injury. In sum, CR2/CR1 play an unanticipated but critical role in the development of a subset of the natural Ab repertoire that has particular importance in the pathogenesis of IR injury.     

Switches, catapults, and chaperones: steady-state kinetic analysis of Hsp70-substrate interactions

Hsp70 chaperones are heterotropic allosteric systems in which ATP and misfolded or aggregated polypeptides are the activating ligands. To gain insight into the mechanism by which ATP and polypeptides regulate Hsp70 chaperone activity, the effect of a short peptide on the K(M) for ATP was analyzed using the Escherichia coli Hsp70 called DnaK. In the absence of peptide, the K(-P)(M) for ATP is 52 +/- 11 nM, whereas this value jumps to 14.6 +/- 1.6 microM in the presence of saturating peptide. This finding supports a mechanism in which ATP binding drives the chaperone in one direction and peptide binding pushes the chaperone back in the opposite direction (and thus increases K(M)), according to ATP + DnaK.P <==> ATP.DnaK.P <==> ATP.DnaK* + P, where ATP.DnaK.P is an intermediate from which competing ATP hydrolysis occurs (ATP.DnaK.P --> ADP.DnaK.P). We show that this branched mechanism can even explain how DnaK hydrolyzes ATP in the absence of peptide and that the true rate constant for DnaK-mediated ATP hydrolysis (k(hy)) in the absence of peptide may be as high as 0.5 s(-)(1) (rather than 5 x 10(-)(4) s(-)(1) as often stated in the literature). What happens is that a conformational equilibrium outcompetes ATP hydrolysis and effectively reduces the concentration of the intermediate by a factor of a thousand, resulting in the following relation: k(cat) = k(hy)/1000 = 5 x 10(-)(4) s(-)(1). How polypeptide substrates and the co-chaperone DnaJ modulate DnaK to achieve its theoretical maximal rate of ATP hydrolysis, which we suggest is 0.5 s(-)(1), is discussed.     

Nucleic acid binding properties and intermediates of HCV core protein multimerization in Pichia pastoris

Little is known about the in vivo assembly pathway or structure of the hepatitis C virus nucleocapsid. In this work the intermediates of HCcAg multimerization in Pichia pastoris cells and the nucleic acid binding properties of structured nucleocapsid-like particles (NLPs) were studied. Extensive cross-linking was observed for HCcAg after glutaraldehyde treatment. Data suggest that HCcAg exists in dimeric forms probably representing P21-P21, P21-P23, and P23-P23 dimers. In addition, the presence of HCcAg species that might represent trimers and multimers was observed. After sucrose equilibrium density gradient purification and nuclease digestion, NLPs were shown to contain both RNA and DNA molecules. Finally, the analysis by electron microscopy indicated that native NLPs were resistant to nuclease treatment. These results indicated that HCcAg assembles through dimers, trimers, and multimers' intermediates into capsids in P. pastoris cells. Assembly of NLPs in its natural environment might confer stability to these particles by adopting a compact structure.