The AMPA receptor subunits GluR-A and GluR-B reciprocally modulate spinal synaptic plasticity and inflammatory pain

Ca(2+)-permeable AMPA receptors are densely expressed in the spinal dorsal horn, but their functional significance in pain processing is not understood. By disrupting the genes encoding GluR-A or GluR-B, we generated mice exhibiting increased or decreased numbers of Ca(2+)-permeable AMPA receptors, respectively. Here, we demonstrate that AMPA receptors are critical determinants of nociceptive plasticity and inflammatory pain. A reduction in the number of Ca(2+)-permeable AMPA receptors and density of AMPA channel currents in spinal neurons of GluR-A-deficient mice is accompanied by a loss of nociceptive plasticity in vitro and a reduction in acute inflammatory hyperalgesia in vivo. In contrast, an increase in spinal Ca(2+)-permeable AMPA receptors in GluR-B-deficient mice facilitated nociceptive plasticity and enhanced long-lasting inflammatory hyperalgesia. Thus, AMPA receptors are not mere determinants of fast synaptic transmission underlying basal pain sensitivity as previously thought, but are critically involved in activity-dependent changes in synaptic processing of nociceptive inputs.     

Additional haplogroups of Toxoplasma gondii out of Africa: population structure and mouse-virulence of strains from Gabon

Background

Toxoplasma gondii is found worldwide, but distribution of its genotypes as well as clinical expression of human toxoplasmosis varies across the continents. Several studies in Europe, North America and South America argued for a role of genotypes in the clinical expression of human toxoplasmosis. Genetic data concerning T. gondii isolates from Africa are scarce and not sufficient to investigate the population structure, a fundamental analysis for a better understanding of distribution, circulation, and transmission.

Methodology/Principal Findings

Seropositive animals originating from urban and rural areas in Gabon were analyzed for T. gondii isolation and genotyping. Sixty-eight isolates, including one mixed infection (69 strains), were obtained by bioassay in mice. Genotyping was performed using length polymorphism of 13 microsatellite markers located on 10 different chromosomes. Results were analyzed in terms of population structure by Bayesian statistical modeling, Neighbor-joining trees reconstruction based on genetic distances, F _ST and linkage disequilibrium. A moderate genetic diversity was detected. Three haplogroups and one single genotype clustered 27 genotypes. The majority of strains belonged to one haplogroup corresponding to the worldwide Type III. The remaining strains were distributed into two haplogroups (Africa 1 and 3) and one single genotype. Mouse virulence at isolation was significantly different between haplogroups. Africa 1 haplogroup was the most virulent.

Conclusion

Africa 1 and 3 haplogroups were proposed as being new major haplogroups of T. gondii circulating in Africa. A possible link with strains circulating in South and Central America is discussed. Analysis of population structure demonstrated a local spread within a rural area and strain circulation between the main cities of the country. This circulation, favored by human activity could lead to genetic exchanges. For the first time, key epidemiological questions were addressed for the West African T. gondii population, using the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological and clinical investigations.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Thermoregulated Expression and Characterization of an NAD(P)H-Dependent 2-Cyclohexen-1-one Reductase in the Plant Pathogenic Bacterium Pseudomonas syringae pv. glycinea

The phytopathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 causes bacterial blight of soybeans and preferably infects its host plant during periods of cold, humid weather conditions. To identify proteins differentially expressed at low temperatures, total cellular protein fractions derived from PG4180.N9 grown at 18 and 28°C were separated by two-dimensional gel electrophoresis. Of several proteins which appeared to be preferentially present at 18°C, a 40-kDa protein with an isoelectric point of approximately 5 revealed significant N-terminal sequence homology to morphinone reductase (MR) of Pseudomonas putida M10. The respective P. syringae gene was isolated from a genomic cosmid library of PG4180, and its nucleotide sequence was determined. It was designated ncr for NAD(P)H-dependent 2-cyclohexen-1-one reductase. Comparison of the 1,083-bp open reading frame with database entries revealed 48% identity and 52% similarity to the MR-encoding morB gene of P. putida M10. The ncr gene was overexpressed in Escherichia coli, and its gene product was used to generate polyclonal antisera. Purified recombinant Ncr protein was enzymatically characterized with NAD(P)H and various morphinone analogs as substrates. So far, only 2-cyclohexen-1-one and 3-penten-2-one were found to be substrates for Ncr. By high-pressure liquid chromatography analysis, flavin mononucleotide could be identified as the noncovalently bound prosthetic group of this enzyme. The distribution of the ncr gene in different Pseudomonas species and various strains of P. syringae was analyzed by PCR and Southern blot hybridization. The results indicated that the ncr gene is widespread among P. syringae pv. glycinea strains but not in other pathovars of P. syringae or in any of the other Pseudomonas strains tested.     

No need to breed for enhanced colonization by arbuscular mycorrhizal fungi to improve low-P adaptation of West African sorghums

Background and aims

Herbaspirillum seropedicae (Hs) Z67 a diazotrophic endophyte was genetically engineered for secretion of 2-keto-D-gluconic acid by heterologous expression of genes for pqq synthesis and gluconate dehydrogenase to study its beneficial effect on plants.

Methods

Two plasmids, pJNK5, containing a 5.1 Kb pqq gene cluster of Acinetobacter calcoaceticus and pJNK6, carrying in addition the Pseudomonas putida KT2440 gluconate dehydrogenase (gad) operon were constructed in pUCPM18Gm^r under Plac promoter. H. seropedicae Z67 transformants were monitored for P and K solubilization, cadmium (Cd) tolerance and rice growth promotion.

Results

Hs (pJNK5) secreted 23.5 mM gluconic acid and Hs (pJNK6) secreted 3.79 mM gluconic acid and 15.8 mM 2-ketogluconic acid respectively. Under aerobic conditions, Hs (pJNK5) and Hs (pJNK6) solubilized 239.7 μM and 457.7 μM P on HEPES rock phosphate and, 76.7 μM and 222.7 μM K on HRPF (feldspar), respectively, in minimal medium containing 50 mM glucose. Under N free minimal medium, similar effects of P and K solubilization were obtained. Hs (pJNK5) and Hs (pJNK6) inoculation increased the biomass, N, P, K content of rice plants (Gujarat – 17). These plants also accumulated 0.73 ng/g PQQ, and had improved growth and tolerance to CdCl₂.

Conclusions

Incorporation of pqq and gad gene clusters in H. seropedicae Z67 imparted additional plant growth promoting traits of P and K solubilization and ability to alleviate Cd toxicity to the host plant.

     

Reduced X-linked diversity in derived populations of house mice

Contrasting patterns of X-linked vs. autosomal diversity may be indicative of the mode of selection operating in natural populations. A number of observations have shown reduced X-linked (or Z-linked) diversity relative to autosomal diversity in various organisms, suggesting a large impact of genetic hitchhiking. However, the relative contribution of other forces such as population bottlenecks, variation in reproductive success of the two sexes, and differential introgression remains unclear. Here, we survey 13 loci, 6 X-linked and 7 autosomal, in natural populations of the house mouse (Mus musculus) subspecies complex. We studied seven populations of three different subspecies, the eastern house mouse M. musculus castaneus, the central house mouse M. m. musculus, and the western house mouse M. m. domesticus, including putatively ancestral and derived populations for each. All populations display lower diversity on the X chromosomes relative to autosomes, and this effect is most pronounced in derived populations. To assess the role of demography, we fit the demographic parameters that gave the highest likelihood of the data using coalescent simulations. We find that the reduction in X-linked diversity is too large to be explained by a simple demographic model in at least two of four derived populations. These observations are also not likely to be explained by differences in reproductive success between males and females. They are consistent with a greater impact of positive selection on the X chromosome, and this is supported by the observation of an elevated K(A) and elevated K(A)/K(S) ratios on the rodent X chromosome. A second contribution may be that the X chromosome less readily introgresses across subspecies boundaries.     

Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Wild-Type Measles Viruses with Non-Standard Genome Lengths

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible.     

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction

Interleukin (IL)-36α is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36α and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36α induces MAP-kinase and NFκB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines.

Methods

To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36α signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36α was tested in murine and human osteoclast assays.

Results

Diseased mice showed an increased expression of IL-36R and IL-36α in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays.

Conclusion

Thus we conclude that IL-36α does not affect the development of inflammatory arthritis.