Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 microM, limiting both purine oxidation and formation of superoxide (O2.). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2. production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.     

Pregnancy initiation in the rhesus macaque: Towards functional manipulation of the maternal-fetal interface

Nonhuman primates provide an important opportunity to define the mechanisms that contribute to the success of early pregnancy. We have focused for several years now on defining the expression of novel placental major histocompatibility complex (MHC) class I molecules. In parallel, we have used reagents against human immune cell markers to characterize the leukocyte population in the decidua and have demonstrated dynamic changes in these cell populations during the first 5 weeks of gestation. The challenge is to identify the possible role(s) of placental MHC class I in modifying/directing the maternal endometrial or systemic immune system in the post-implantation period. Foremost among the challenges is the difficulty in modifying placental function. In the instance of trophoblast surface proteins, passive immunization studies are feasible, although limitations include the empirical nature of this approach, as well as the inability to modify intracellular function. We have shown that using lentiviral vectors to effect preimplantation gene transfer for transgene expression in the placenta is not only feasible, but of good efficiency. In addition to transgene overexpression, robust approaches for knocking down/knocking out placental gene expression are essential. Recent developments in RNA interference approaches may allow "transient knockout" experiments. While the rhesus monkey has been our model of choice, currently there are limitations in the number of available female rhesus monkeys of reproductive age for research in early pregnancy. It is critical that the technologies for advanced study move forward in other species. The baboon has been used significantly in reproductive tract biology and early pregnancy research and important models have been developed for manipulation of the maternal-fetal interface. Additional characterization of other species, such as the cynomolgus and African green (vervet) monkey is critical. Given the limitations on antigen recognition when using human reagents, we also propose that the development of panels of primate-specific anti-leukocyte antibodies is essential for moving forward nonhuman primate reproductive research.     

Response from Jeffrey I. Gordon et al.: Commensal bacteria make a difference

The importance of the gut microbiota has been recognized since the days of Pasteur. What makes today different from yesterday, and tomorrow so exciting, is that we now have the tools to identify the molecular mechanisms that regulate assembly of the microbiota and determine how its components affect postnatal mammalian development and adult physiology.     

Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-alpha

As the first immune cells to infiltrate the nervous system after traumatic PNS and CNS injury, neutrophils (polymorphonuclear leukocytes, PMNs) may promote injury by releasing toxic soluble factors that may affect neuronal survival. Direct neurotoxicity of matrix metalloproteinases (MMPs), reactive oxygen species (ROS), and cytokines released by PMNs was investigated by culturing dorsal root ganglion (DRG) cells with PMN-conditioned media containing MMP inhibitor (GM6001), ROS scavengers, or tumor necrosis factor alphaR (TNF-alphaR) neutralizing antibody. Although DRGs exposed to PMN-conditioned media had 53% fewer surviving neurons than controls, neuronal cell loss was prevented by GM6001 (20 micromol/L), catalase (1000 U/mL), or TNF-alphaR neutralizing antibody (1.5 microg/mL), elevating survival to 77%, 94%, and 95%, respectively. In accordance with protection by GM6001, conditioned media collected from MMP-9 null PMNs was less neurotoxic than that collected from wild-type PMNs. Additionally, MMP inhibition reduced PMN-derived ROS; removal of ROS reduced PMN-derived MMP-9 activity; and TNF-alpha inhibition reduced both PMN-derived MMP-9 activity and ROS in PMN cultures. Our data provide the first direct evidence that PMN-driven neurotoxicity is dependent on MMPs, ROS, and TNF-alpha, and that these factors may regulate PMN release of these soluble factors or interact with one another to mediate PMN-driven neurotoxicity.     

Human enteropathogen load in activated sewage sludge and corresponding sewage sludge end products

This study demonstrated a significant reduction in the concentrations of Cryptosporidium parvum and Cryptosporidium hominis oocysts, Giardia lamblia cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.     

Isolate Identity Determines Plant Tolerance to Pathogen Attack in Assembled Mycorrhizal Communities

Arbuscular mycorrhizal fungi (AMF) are widespread soil microorganisms that associate mutualistically with plant hosts. AMF receive photosynthates from the host in return for various benefits. One of such benefits is in the form of enhanced pathogen tolerance. However, this aspect of the symbiosis has been understudied compared to effects on plant growth and its ability to acquire nutrients. While it is known that increased AMF species richness positively correlates with plant productivity, the relationship between AMF diversity and host responses to pathogen attack remains obscure. The objective of this study was to test whether AMF isolates can differentially attenuate the deleterious effects of a root pathogen on plant growth, whether the richest assemblage of AMF isolates provides the most tolerance against the pathogen, and whether AMF-induced changes to root architecture serve as a mechanism for improved plant disease tolerance. In a growth chamber study, we exposed the plant oxeye daisy (Leucanthemum vulgare) to all combinations of three AMF isolates and to the plant root pathogen Rhizoctonia solani. We found that the pathogen caused an 81% reduction in shoot and a 70% reduction in root biomass. AMF significantly reduced the highly deleterious effect of the pathogen. Mycorrhizal plants infected with the pathogen produced 91% more dry shoot biomass and 72% more dry root biomass relative to plants solely infected with R. solani. AMF isolate identity was a better predictor of AMF-mediated host tolerance to the pathogen than AMF richness. However, the enhanced tolerance response did not result from AMF-mediated changes to root architecture. Our data indicate that AMF communities can play a major role in alleviating host pathogen attack but this depends primarily on the capacity of individual AMF isolates to provide this benefit.     

Nonhuman primate transgenesis: progress and prospects

The nonhuman primate is used extensively in biomedical research owing to its close similarities to human physiology and human disease pathophysiology. Recently, several groups have initiated efforts to genetically manipulate nonhuman primates to address complex questions concerning primate-specific development and physiological adaptation. Primates pose unique challenges to transgenesis and, although this field is still in its infancy, the potential for obtaining new insights into primate physiology and gene function is unprecedented. This review focuses on the methods and potential applications of genetically altered nonhuman primates in biomedical research.     

Characterization and Chromosomal Localization of the Gene for Human Rhodopsin Kinase

G-protein-dependent receptor kinases (GRKs) play a key role in the adaptation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid desensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the structure and mechanism of GRKs in human photoreceptors, we have isolated and characterized cDNA and genomic clones derived from the human RK locus using a bovine rhodopsin kinase cDNA fragment as a probe. The RK locus, assigned to chromosome 13 band q34, is composed of seven exons that encode a protein 92% identical in amino acid sequence to bovine rhodopsin kinase. The marked difference between the structure of this gene and that of another recently cloned human GRK gene suggests the existence of a wide evolutionary gap between members of the GRK gene family.