Mycotoxin fumonisin B1 increases intestinal colonization by pathogenic Escherichia coli in pigs

Fumonisin B(1) (FB(1)) is a mycotoxin that commonly occurs in maize. FB(1) causes a variety of toxic effects in different animal species and has been implicated as a contributing factor of esophageal cancers in humans. In the present study, we examined the effect of dietary exposure to FB(1) on intestinal colonization by pathogenic Escherichia coli associated with extraintestinal infection. Three-week-old weaned pigs were given FB(1) by gavage as a crude extract or as a purified toxin at a dose of 0.5 mg/kg of body weight daily for 6 days. On the last day of the toxin treatment, the pigs were orally inoculated with an extraintestinal pathogenic E. coli strain. All animals were euthanized 24 h later, necropsies were performed, and tissues were taken for bacterial counts and light microscopic examination. Ingestion of FB(1) had only a minimal effect on animal weight gain, did not cause any macroscopic or microscopic lesions, and did not change the plasma biochemical profile. However, colonization of the small and large intestines by an extraintestinal pathogenic E. coli strain was significantly increased. Our results show that FB(1) is a predisposing factor to infectious disease and that the pig can be used as a model for the study of the consequences of ingesting mycotoxin-contaminated food.     

Relationship between SU subdomains that regulate the receptor-mediated transition from the native (fusion-inhibited) to the fusion-active conformation of the murine leukemia virus glycoprotein

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in cis or in trans, between the receptor-activated RBD and a median segment of the freed C-terminal domain.     

(2-amino-phenyl)-amides of omega-substituted alkanoic acids as new histone deacetylase inhibitors

A variety of omega-substituted alkanoic acid (2-amino-phenyl)-amides were designed and synthesized. These compounds were shown to inhibit recombinant human histone deacetylases (HDACs) with IC(50) values in the low micromolar range and induce hyperacetylation of histones in whole cells. They induced expression of p21WAF1/Cip1 and caused cell-cycle arrest in human cancer cells. Compounds in this class showed efficacy in human tumor xenograft models.     

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmoniers algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.     

An InCytes from MBC Selection: Mice Lacking Mannose 6-Phosphate Uncovering Enzyme Activity Have a Milder Phenotype than Mice Deficient for N-Acetylglucosamine-1-Phosphotransferase Activity

The mannose 6-phosphate (Man-6-P) lysosomal targeting signal on acid hydrolases is synthesized by the sequential action of uridine 5′-diphosphate-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-1-phosphotransferase) and GlcNAc-1-phosphodiester α-N-acetylglucosaminidase (“uncovering enzyme” or UCE). Mutations in the two genes that encode GlcNAc-1-phosphotransferase give rise to lysosomal storage diseases (mucolipidosis type II and III), whereas no pathological conditions have been associated with the loss of UCE activity. To analyze the consequences of UCE deficiency, the UCE gene was inactivated via insertional mutagenesis in mice. The UCE −/− mice were viable, grew normally and lacked detectable histologic abnormalities. However, the plasma levels of six acid hydrolases were elevated 1.6- to 5.4-fold over wild-type levels. These values underestimate the degree of hydrolase hypersecretion as these enzymes were rapidly cleared from the plasma by the mannose receptor. The secreted hydrolases contained GlcNAc-P-Man diesters, exhibited a decreased affinity for the cation-independent mannose 6-phosphate receptor and failed to bind to the cation-dependent mannose 6-phosphate receptor. These data demonstrate that UCE accounts for all the uncovering activity in the Golgi. We propose that in the absence of UCE, the weak binding of the acid hydrolases to the cation-independent mannose 6-phosphate receptor allows sufficient sorting to lysosomes to prevent the tissue abnormalities seen with GlcNAc-1-phosphotranferase deficiency.     

CD4+CD28null T cells in autoimmune disease: pathogenic features and decreased susceptibility to immunoregulation

To determine the role of expanded CD4(+)CD28(null) T cells in multiple sclerosis and rheumatoid arthritis pathology, these cells were phenotypically characterized and their Ag reactivity was studied. FACS analysis confirmed that CD4(+)CD28(null) T cells are terminally differentiated effector memory cells. In addition, they express phenotypic markers that indicate their capacity to infiltrate into tissues and cause tissue damage. Whereas no reactivity to the candidate autoantigens myelin basic protein and collagen type II was observed within the CD4(+)CD28(null) T cell subset, CMV reactivity was prominent in four of four HC, four of four rheumatoid arthritis patients, and three of four multiple sclerosis patients. The level of the CMV-induced proliferative response was found to be related to the clonal diversity of the response. Interestingly, our results illustrate that CD4(+)CD28(null) T cells are not susceptible to the suppressive actions of CD4(+)CD25(+) regulatory T cells. In conclusion, this study provides several indications for a role of CD4(+)CD28(null) T cells in autoimmune pathology. CD4(+)CD28(null) T cells display pathogenic features, fill up immunological space, and are less susceptible to regulatory mechanisms. However, based on their low reactivity to the autoantigens tested in this study, CD4(+)CD28(null) T cells most likely do not play a direct autoaggressive role in autoimmune disease.     

Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception

Transgenic tomato [Lycopersicon esculentum (=Solanum lycopersicum)] lines overexpressing tomato PHYA, PHYB1, or PHYB2, under control of the constitutive double-35S promoter from cauliflower mosaic virus (CaMV) have been generated to test the level of saturation in individual phytochrome-signalling pathways in tomato. Western blot analysis confirmed the elevated phytochrome protein levels in dark-grown seedlings of the respective PHY overexpressing (PHYOE) lines. Exposure to 4 h of red light resulted in a decrease in phytochrome A protein level in the PHYAOE lines, indicating that the chromophore availability is not limiting for assembly into holoprotein and that the excess of phytochrome A protein is also targeted for light-regulated destruction. The elongation and anthocyanin accumulation responses of plants grown under white light, red light, far-red light, and end-of-day far-red light were used for characterization of selected PHYOE lines. In addition, the anthocyanin accumulation response to different fluence rates of red light of 4-d-old dark-grown seedlings was studied. The elevated levels of phyA in the PHYAOE lines had little effect on seedling and adult plant phenotype. Both PHYAOE in the phyA mutant background and PHYB2OE in the double-mutant background rescued the mutant phenotype, proving that expression of the transgene results in biologically active phytochrome. The PHYB1OE lines showed mild effects on the inhibition of stem elongation and anthocyanin accumulation and little or no effect on the red light high irradiance response. By contrast, the PHYB2OE lines showed a strong inhibition of elongation, enhancement of anthocyanin accumulation, and a strong amplification of the red light high irradiance response.     

Rapid coupling of Surface Plasmon Resonance (SPR and SPRi) and ProteinChip based mass spectrometry for the identification of proteins in nucleoprotein interactions

We compared coupling approaches of SPR to LC-MS and ProteinChip-based mass spectrometry (SELDI) as a means of identifying proteins captured on DNA surfaces. The approach we outline has the potential to allow multiple, quantitative analysis of macromolecular interactions followed by rapid mass spectrometry identification of retained material.