Mineralization of 2,4,6-trinitrophenol (picric acid): Characterization and phylogenetic identification of microbial strains

Four bacterial strains that use picric acid as their sole carbon and energy source were isolated. Mineralization of¹⁴C-UL-picric acid showed that up to 65% of the radioactivity was released as¹⁴CO₂. HPLC and UV/Vis spectral analyses indicated complete degradation of picric acid by these organisms. HPLC and LC/MS analyses showed transient formation of 2,4-dinitrophenol during picric acid degradation. Degradation of picric acid was concomitant with stoichiometric release of three moles of nitrite per mole of picric acid. The four picric acid degraders were identified as close relatives ofNocardioides simplex (ATCC 6946) based on their small subunit (16S) rRNA gene sequences.This is contribution 7167 from Central Research & Development, Dupont Co, Wilmington, DE 19880, USA     

Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

Comparison of salicylate and D-phenylalanine for detection of hydroxyl radicals in chemical and biological reactions

Summary

Hydroxylation of salicylate and D-phenylalanine was measured to test the usefulness of these compounds for hydroxyl radical (HO^?) detection in chemical and biological systems. When HO^? were produced by the photolytic decomposition of hydrogen peroxide, nearly equal amounts of 2,5- and 2,3-dihydroxybenzoic acid (DHBA) were produced from salicylate, with catechol as a minor product. In the photolytic reaction, nearly equal concentrations of p-,m-, and o-tyrosine were formed from D-phenylalanine. When salicylate or D-phenylalanine was present with Fenton reagents or in iron(II) autoxidation systems, the relative proportions of hydroxylated products were similar to those observed after photolysis, although less total products were usually detected. In contrast, when similar experiments were conducted with isolated hepatic microsomes and perfused livers, 2,5-DHBA was the primary product from salicylate, and p-tyrosine was the major product from D-phenylalanine. Cytochrome P-450 enzymes can hydroxylate salicylate to produce 2,5-DHBA, and it is likely that phenylalanine hydroxylase produces most of the p-tyrosine detected in hepatic tissues. Thus, although both salicylate and D-phenylalanine are useful probes for hydroxyl radical formation in chemical systems, hydroxylated products formed from enzymatic reactions complicate interpretation of data from both compounds in vivo.     

TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation

In the adult mammalian brain, neural stem cells in the subventricular zone continuously generate new neurons for the olfactory bulb. Cell fate commitment in these adult neural stem cells is regulated by cell fate-determining proteins. Here, we show that the cell fate-determinant TRIM32 is upregulated during differentiation of adult neural stem cells into olfactory bulb neurons. We further demonstrate that TRIM32 is necessary for the correct induction of neuronal differentiation in these cells. In the absence of TRIM32, neuroblasts differentiate slower and show gene expression profiles that are characteristic of immature cells. Interestingly, TRIM32 deficiency induces more neural progenitor cell proliferation and less cell death. Both effects accumulate in an overproduction of adult-generated olfactory bulb neurons of TRIM32 knockout mice. These results highlight the function of the cell fate-determinant TRIM32 for a balanced activity of the adult neurogenesis process.     

Levels of IL-17F and IL-33 correlate with HLA-DR activation in clinical-grade human bone marrow–derived multipotent mesenchymal stromal cell expansion cultures

Background aims

Multipotent mesenchymal stromal cell (MSC)-based medicines are extensively investigated for use in regenerative medicine and immunotherapy applications. The International Society for Cell and Gene Therapy (ISCT) proposed a panel of cell surface molecules for MSC identification that includes human leukocyte antigen (HLA)-DR as a negative marker. However, its expression is largely unpredictable despite production under tightly controlled conditions and compliance with current Good Manufacturing Practices. Herein, we report the frequency of HLA-DR expression in 81 batches of clinical grade bone marrow (BM)-derived MSCs and investigated its impact on cell attributes and culture environment.

Pathogen fitness components and genotypes differ in their sensitivity to nutrient and temperature variation in a wild plant-pathogen association

Understanding processes maintaining variation in pathogen life-history stages affecting infectivity and reproduction is a key challenge in evolutionary ecology. Models of host-parasite coevolution are based on the assumption that genetic variation for host-parasite interactions is a significant cause of variation in infection, and that variation in environmental conditions does not overwhelm the genetic basis. However, surprisingly little is known about the stability of genotype-genotype interactions under variable environmental conditions. Here, using a naturally occurring plant-pathogen interaction, I tested whether the two distinct aspects of the infection process - infectivity and transmission potential - vary over realistic nutrient and temperature gradients. I show that the initial pathogen infectivity and host resistance responses are robust over the environmental gradients. However, for compatible responses there were striking differences in how different pathogen life-history stages and host and pathogen genotypes responded to environmental variation. For some pathogen genotypes even slight changes in temperature arrested spore production, rendering the developing infection ineffectual. The response of pathogen genotypes to environmental gradients varied in magnitude and even direction, so that their rankings changed across the abiotic gradients. Hence, the variable environment of spatially structured host-parasite interactions may strongly influence the maintenance of polymorphism in pathogen life-history stages governing transmission, whereas evolutionary trajectories of infectivity may be unaffected by the surrounding environment.     

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

The role of extracellular α-synuclein (α-syn) in the initiation and the spreading of neurodegeneration in Parkinson''s disease (PD) has been studied extensively over the past 10 years. However, the nature of the α-syn toxic species and the molecular mechanisms by which they may contribute to neuronal cell loss remain controversial. In this study, we show that fully characterized recombinant monomeric, fibrillar or stabilized forms of oligomeric α-syn do not trigger significant cell death when added individually to neuroblastoma cell lines. However, a mixture of preformed fibrils (PFFs) with monomeric α-syn becomes toxic under conditions that promote their growth and amyloid formation. In hippocampal primary neurons and ex vivo hippocampal slice cultures, α-syn PFFs are capable of inducing a moderate toxicity over time that is greatly exacerbated upon promoting fibril growth by addition of monomeric α-syn. The causal relationship between α-syn aggregation and cellular toxicity was further investigated by assessing the effect of inhibiting fibrillization on α-syn-induced cell death. Remarkably, our data show that blocking fibril growth by treatment with known pharmacological inhibitor of α-syn fibrillization (Tolcapone) or replacing monomeric α-syn by monomeric β-synuclein in α-syn mixture composition prevent α-syn-induced toxicity in both neuroblastoma cell lines and hippocampal primary neurons. We demonstrate that exogenously added α-syn fibrils bind to the plasma membrane and serve as nucleation sites for the formation of α-syn fibrils and promote the accumulation and internalization of these aggregates that in turn activate both the extrinsic and intrinsic apoptotic cell death pathways in our cellular models. Our results support the hypothesis that ongoing aggregation and fibrillization of extracellular α-syn play central roles in α-syn extracellular toxicity, and suggest that inhibiting fibril growth and seeding capacity constitute a viable strategy for protecting against α-syn-induced toxicity and slowing the progression of neurodegeneration in PD and other synucleinopathies.The discovery of α-synuclein (α-syn) as the main component of Lewy bodies (LBs) and the identification of gene duplication and missense mutations in the α-syn gene in some familial forms of Parkinson''s disease (PD) have reinforced the central role of α-syn in the etiology of both sporadic and familial cases of PD.¹ Nevertheless, the relationship between α-syn aggregation and neurodegeneration in PD remains elusive.²A possible role for extracellular α-syn in the pathogenicity of PD emerged from the observation that newly grafted neurons in PD patients exhibit α-syn pathology similar to that of neighboring diseased cells.^{3, 4} Despite the consensus that α-syn is mainly an intracellular protein, α-syn has been detected in the cerebrospinal fluid under both pathological and healthy conditions.⁵ In addition, in vivo rodent models and cellular studies have shown that monomers⁶ and aggregated forms^{6, 7} of α-syn are secreted into the extracellular space via several mechanisms,^{7, 8} including the nonclassical endoplasmic reticulum/Golgi-independent exocytosis⁸ or the exosomal route,^{9, 10} and are then internalized by neighboring cells.⁷ This suggests that extracellular α-syn may play a critical role in the spreading of α-syn pathology throughout the brain and contributes to PD progression.Additional evidence for a causal role of extracellular α-syn in PD come from in vivo studies and cell culture models: (1) intracranial injections of pathological forms of α-syn, isolated from LBs or old mice, as well as recombinant α-syn fibrils, were shown to nucleate further α-syn aggregation, pathology spreading and trigger neurodegeneration in vivo in wild-type (WT) or transgenic mice^{11, 12, 13, 14} and rhesus monkeys;¹⁵ (2) recombinant extracellular α-syn aggregates are internalized in cultured cells and seed the aggregation of endogenous α-syn;^{12, 16, 17, 18} and (3) extracellular α-syn activates microglia that initiates or enhances nigral neurodegeneration.^{19, 20, 21} Although the toxic effects of exogenous recombinant α-syn have been thoroughly investigated in different cellular models,^{7, 17, 18, 22, 23, 24, 25} the relative contribution of monomeric, oligomeric and fibrillar forms of α-syn to the overall toxicity remains controversial. Therefore, the identification of toxic α-syn species and the molecular mechanisms by which they contribute to neurodegeneration is required to better understand how extracellular α-syn contributes to PD pathogenesis and to develop novel strategies for the diagnosis and treatment of PD and other synucleinopathies. In our study we explored the relationship between neurotoxicity and the aggregation state or amyloid formation propensity of α-syn in various cellular models.     

MC1R-dependent, melanin-based colour polymorphism is associated with cell-mediated response in the Eleonora's falcon

Colour polymorphism in vertebrates is usually under genetic control and may be associated with variation in physiological traits. The melanocortin 1 receptor (Mc1r) has been involved repeatedly in melanin-based pigmentation but it was thought to have few other physiological effects. However, recent pharmacological studies suggest that MC1R could regulate the aspects of immunity. We investigated whether variation at Mc1r underpins plumage colouration in the Eleonora's falcon. We also examined whether nestlings of the different morphs differed in their inflammatory response induced by phytohemagglutinin (PHA). Variation in colouration was due to a deletion of four amino acids at the Mc1r gene. Cellular immune response was morph specific. In males, but not in females, dark nestling mounted a lower PHA response than pale ones. Although correlative, our results raise the neglected possibility that MC1R has pleiotropic effects, suggesting a potential role of immune capacity and pathogen pressure on the maintenance of colour polymorphism in this species.