Suppression of cold ischemic injury in stored kidneys by the antimicrobial peptide bactenecin

BACKGROUND: Cold ischemic injury plays an important role in short- and long-term function of kidneys after transplant. Antimicrobial peptides have not previously been studied for their impact on cold ischemia in transplanted kidneys. METHODS: Bactenecin (L- and D-forms) was added to University of Wisconsin (UW) preservation solution for 3-day cold storage of dog kidneys. Effects on membrane permeability were studied in synthetic liposomes and in kidney cortex tissue slices. The role of bactenecin as a tissue mitogen and direct cytoskeletal stabilizer were studied with cultured cells and in vitro. RESULTS: Bactenecin (both L- and D- forms) resulted in significant decreases in postoperative serum creatinine and time required for return of creatinine to the normal range showing the effect was independent of chirality. Bactenecin permeabilized synthetic liposomes and altered kidney cortex tissue slice membrane permeability characteristics, irrespective of chirality. Neither did bactenecin act as a mitogen for either primary renal tubule or Madin-Darby canine kidney (MDCK) cells stored in UW solution, nor did it appear to directly affect cytoskeletal dynamics. CONCLUSIONS: These results show that the antimicrobial peptide bactenecin can improve the quality of static cold storage of kidneys. The mechanism of its action is independent of receptor binding and does not appear to involve either an effect on the cytoskeleton or via activity as a mitogen. Current evidence best supports the hypothesis that bactenecin protects against cold ischemic injury by a controlled permeabilization of the membranes of the kidney during cold storage.     

Multiple loci and epistases control genetic variation for seed dormancy in weedy rice (Oryza sativa)

Weedy rice has much stronger seed dormancy than cultivated rice. A wild-like weedy strain SS18-2 was selected to investigate the genetic architecture underlying seed dormancy, a critical adaptive trait in plants. A framework genetic map covering the rice genome was constructed on the basis of 156 BC(1) [EM93-1 (nondormant breeding line)//EM93-1/SS18-2] individuals. The mapping population was replicated using a split-tiller technique to control and better estimate the environmental variation. Dormancy was determined by germination of seeds after 1, 11, and 21 days of after-ripening (DAR). Six dormancy QTL, designated as qSD(S)-4, -6, -7-1, -7-2, -8, and -12, were identified. The locus qSD(S)-7-1 was tightly linked to the red pericarp color gene Rc. A QTL x DAR interaction was detected for qSD(S)-12, the locus with the largest main effect at 1, 11, and 21 DAR (R(2) = 0.14, 0.24, and 0.20, respectively). Two, three, and four orders of epistases were detected with four, six, and six QTL, respectively. The higher-order epistases strongly suggest the presence of genetically complex networks in the regulation of variation for seed dormancy in natural populations and make it critical to select for a favorable combination of alleles at multiple loci in positional cloning of a target dormancy gene.     

The structure of the 1L-myo-inositol-1-phosphate synthase-NAD+-2-deoxy-D-glucitol 6-(E)-vinylhomophosphonate complex demands a revision of the enzyme mechanism

1l-myo-inositol 1-phosphate (MIP) synthase catalyzes the conversion of d-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, the first and rate-limiting step in the biosynthesis of all inositol-containing compounds. It involves an oxidation, enolization, intramolecular aldol cyclization, and reduction. Here we present the structure of MIP synthase in complex with NAD(+) and a high-affinity inhibitor, 2-deoxy-d-glucitol 6-(E)-vinylhomophosphonate. This structure reveals interactions between the enzyme active site residues and the inhibitor that are significantly different from that proposed for 2-deoxy-d-glucitol 6-phosphate in the previously published structure of MIP synthase-NAD(+)-2-deoxy-d-glucitol 6-phosphate. There are several other conformational changes in NAD(+) and the enzyme active site as well. Based on the new structural data, we propose a new and completely different mechanism for MIP synthase.     

Identification and H2O2 sensitivity of the major constitutive MAPK phosphatase from rat brain

The present study examined in subcellular fractions from rat brain the nature and sensitivity to hydrogen peroxide of constitutively expressed mitogen-activated protein kinase (MAPK) phosphatase activity. MAPK phosphatase activity was defined as the activity directed towards a dual-phosphorylated (pT/pY) peptide corresponding to the activation domain of the extracellular-regulated kinase (ERK) subtype of the MAPKs. The use of phosphatase inhibitors and biochemical analyses demonstrate that the MAPK phosphatase activity, which was highest in the microsomal membrane and soluble fractions, was attributable mainly, if not entirely, to protein phosphatase 2A (PP2A). Moreover, hydrogen peroxide (in the absence and presence of reduced glutathione) and glutathione disulfide inhibited the MAPK phosphatase activity by a dithiothreitol-reversible mechanism. These results provide direct support for mounting evidence that PP2A is a major regulator of MAPK phosphorylation in brain and suggest that inhibition of PP2A activity via reversible oxidation of a cysteine thiol(s) may underlie at least in part the activation of MAPKs occurring in response to hydrogen peroxide and oxidative stress.     

An Improved Phenylarsine Oxide-Affinity Method Identifies Triose Phosphate Isomerase as a Candidate Redox Receptor Protein

Reversible oxidation on proteins of vicinal thiols to form intraprotein disulfides is believed to be an important means by which redox sensitivity is conferred on cellular signaling and metabolism. Affinity chromatography using immobilized phenylarsine oxide (PAO), which binds preferentially to vicinal thiols over monothiols, has been used in very limited studies to isolate the fraction of cellular proteins that exhibit reversible oxidation of vicinal thiols to presumed disulfide bonds. A challenge to the use of PAO-affinity chromatography for isolation of readily oxidizable vicinal thiol proteins (VTPs) has been the lack of a disulfide reducing agent that reverses oxidation of the PAO-binding protein thiols and maintains these in the reduced state necessary to bind PAO but does not also compete with the VTPs for binding to the immobilized PAO. The present study demonstrates that the capture from a detergent-soluble rat brain extract of VTPs by PAO-affinity chromatography was improved greatly by use of the reducing agent tris(2-carboxyethyl)-phosphine which, unlike more traditional disulfide-reducing agents, does not contain a thiol group. Moreover, we show that, while a substantial fraction of total brain proteins contain PAO-binding thiols, only a fraction of these were readily and reversibly oxidized. The two most abundant of these redox-active proteins were identified as albumin and triose phosphate isomerase (TPI). We propose that TPI is a candidate intracellular redox receptor protein. The improved PAO-affinity method detailed here should enable the discovery of lower abundance novel redox-active regulatory proteins.     

Differences in the Transmissibility of Two Anaplasma phagocytophilum Strains by the North American Tick Vector Species, Ixodes Pacificus and Ixodes Scapularis (Acari: Ixodidae)

The etiologic agent of granulocytic anaplasmosis, Anaplasma phagocytophilum, has a circum-global distribution within the northern hemisphere and shows a host species predilection that varies by the geographic region in which the disease is found. Adaptation by the bacterium to a host species potentially contributes to the variation found worldwide but this is confounded by the bacterium's relationship with its tick vectors, all of which belong to the Ixodes ricinus group. We tested the hypothesis that tick vector species collected from geographic regions sympatric with particular A. phagocytophilum strains will show evidence of a higher degree of vector competence than will tick species and allopatric A. phagocytophilum strains. A reciprocal cross-transmission experiment was performed using an eastern and a western North American strain of A. phagocytophilum (Webster and MRK, respectively) and the two tick species, I. scapularis and I. pacificus, most commonly associated with human and animal transmission of the bacteria in the United States. The western tick, I. pacificus, showed a significantly higher vector competence for A. phagocytophilum than I. scapularis and the eastern isolate, Webster, was more transmissible than its western counterpart, MRK. These results indicate that geographic variation in host susceptibility to A. phagocytophilum strains may play a more important role in the epidemiology of granulocytic anaplasmosis than does the competence of its tick vectors to transmit the pathogen.     

ATP binding cassette transporter G1 deletion induces IL-17-dependent dysregulation of pulmonary adaptive immunity

Mice with genetic deletion of the cholesterol transporter ATP binding cassette G1 (ABCG1) have pulmonary lipidosis and enhanced innate immune responses in the airway. Whether ABCG1 regulates adaptive immune responses to the environment is unknown. To this end, Abcg1(+/+) and Abcg1(-/-) mice were sensitized to OVA via the airway using low-dose LPS as an adjuvant, and then challenged with OVA aerosol. Naive Abcg1(-/-) mice displayed increased B cells, CD4(+) T cells, CD8(+) T cells, and dendritic cells (DCs) in lung and lung-draining mediastinal lymph nodes, with lung CD11b(+) DCs displaying increased CD80 and CD86. Upon allergen sensitization and challenge, the Abcg1(-/-) airway, compared with Abcg1(+/+), displayed reduced Th2 responses (IL-4, IL-5, eosinophils), increased neutrophils and IL-17, but equivalent airway hyperresponsiveness. Reduced Th2 responses were also found using standard i.p. OVA sensitization with aluminum hydroxide adjuvant. Mediastinal lymph nodes from airway-sensitized Abcg1(-/-) mice produced reduced IL-5 upon ex vivo OVA challenge. Abcg1(-/-) CD4(+) T cells displayed normal ex vivo differentiation, whereas Abcg1(-/-) DCs were found paradoxically to promote Th2 polarization. Th17 cells, IL-17(+) γδT cells, and IL-17(+) neutrophils were all increased in Abcg1(-/-) lungs, suggesting Th17 and non-Th17 sources of IL-17 excess. Neutralization of IL-17 prior to challenge normalized eosinophils and reduced neutrophilia in the Abcg1(-/-) airway. We conclude that Abcg1(-/-) mice display IL-17-mediated suppression of eosinophilia and enhancement of neutrophilia in the airway following allergen sensitization and challenge. These findings identify ABCG1 as a novel integrator of cholesterol homeostasis and adaptive immune programs.     

Nutritional limitation and resistance to opportunistic Aspergillus parasites in honey bee larvae

Honey bees are threatened by land use changes which reduce the availability and diversity of pollen and nectar resources. There is concern that poor nutrition may be involved in recent population declines, either directly or due to indirect effects on immunocompetence. The larval stage is likely to be the most vulnerable to a poor diet, but the effects of larval nutrition on the disease susceptibility of bees are not well known. In this study we used laboratory-reared honey bee larvae to investigate the effects of diet quality on disease susceptibility to the opportunistic fungal parasites Aspergillus flavus, Aspergillus phoenicis and A. fumigatus. Larvae fed on a nutritionally poor diet were found to be significantly more susceptible to A. fumigatus. Larval resistance to A. fumigatus was enhanced by feeding with a diet supplemented with either dandelion or polyfloral pollens. This indicates that dandelion and polyfloral pollens contain elements that enhance resistance to this fungal disease, illustrating an interaction between nutrition and parasitism and emphasising the benefit of diverse floral resources in the environment to maintain honey bee health.     

Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes

Rapid development of next generation sequencing (NGS) technologies in recent years has made whole genome sequencing of bacterial genomes widely accessible. However, it is often unnecessary or not feasible to sequence the whole genome for most applications of genetic analyses in bacteria. Selectively capturing defined genomic regions followed by NGS analysis could be a promising approach for high-resolution molecular typing of a large set of strains. In this study, we describe a novel and straightforward PCR-based target-capturing method, hairpin-primed multiplex amplification (HPMA), which allows for simultaneous amplification of numerous target genes. To test the feasibility of NGS-based strain typing using HPMA, 20 target gene sequences were simultaneously amplified with barcode tagging in each of 41 Salmonella strains. The amplicons were then pooled and analyzed by 454 pyrosequencing. Analysis of the sequence data, as an extension of multilocus sequence typing (MLST), demonstrated the utility and potential of this novel typing method, MLST-seq, as a high-resolution strain typing method. With the rapidly increasing sequencing capacity of NGS, MLST-seq or its variations using different target enrichment methods can be expected to become a high-resolution typing method in the near future for high-throughput analysis of a large collection of bacterial strains.