Genomic selection for spot blotch in bread wheat breeding panels, full-sibs and half-sibs and index-based selection for spot blotch, heading and plant height.

KEY MESSAGE: Genomic selection is a promising tool to select for spot blotch resistance and index-based selection can simultaneously select for spot blotch resistance, heading and plant height. A major biotic stress challenging bread wheat production in regions characterized by humid and warm weather is spot blotch caused by the fungus Bipolaris sorokiniana. Since genomic selection (GS) is a promising selection tool, we evaluated its potential for spot blotch in seven breeding panels comprising 6736 advanced lines from the International Maize and Wheat Improvement Center. Our results indicated moderately high mean genomic prediction accuracies of 0.53 and 0.40 within and across breeding panels, respectively which were on average 177.6% and 60.4% higher than the mean accuracies from fixed effects models using selected spot blotch loci. Genomic prediction was also evaluated in full-sibs and half-sibs panels and sibs were predicted with the highest mean accuracy (0.63) from a composite training population with random full-sibs and half-sibs. The mean accuracies when full-sibs were predicted from other full-sibs within families and when full-sibs panels were predicted from other half-sibs panels were 0.47 and 0.44, respectively. Comparison of GS with phenotypic selection (PS) of the top 10% of resistant lines suggested that GS could be an ideal tool to discard susceptible lines, as greater than 90% of the susceptible lines discarded by PS were also discarded by GS. We have also reported the evaluation of selection indices to simultaneously select non-late and non-tall genotypes with low spot blotch phenotypic values and genomic-estimated breeding values. Overall, this study demonstrates the potential of integrating GS and index-based selection for improving spot blotch resistance in bread wheat.

Pyramiding of genes for grain protein content, grain quality, and rust resistance in eleven Indian bread wheat cultivars: a multi-institutional effort.

Improvement of grain protein content (GPC), loaf volume, and resistance to rusts was achieved in 11 Indian wheat cultivars that are widely grown in four different agro-climatic zones of India. This involved use of marker-assisted backcross breeding (MABB) for introgression and pyramiding of the following genes: (i) the high GPC gene Gpc-B1; (ii) HMW glutenin subunits 5 + 10 at Glu-D1 loci, and (iii) rust resistance genes, Yr36, Yr15, Lr24, and Sr24. GPC increased by 0.8 to 3.3%, although high GPC was generally associated with yield penalty. Further selection among high GPC lines allowed identification of progenies with higher GPC associated with improvement in 1000-grain weight and grain yield in the backgrounds of the following four cultivars: NI5439, UP2338, UP2382, and HUW468. The high GPC progenies (derived from NI5439) were also improved for grain quality using HMW glutenin subunits 5 + 10 at Glu-D1 loci. Similarly, progenies combining high GPC and rust resistance were obtained in the backgrounds of following five cultivars: Lok1, HD2967, PBW550, PBW621, and DBW1. The improved pre-bred lines developed following multi-institutional effort should prove a valuable source for the development of cultivars with improved nutritional quality and rust resistance in the ongoing wheat breeding programmes. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01277-w.

Dissecting Quantitative Trait Loci for Spot Blotch Resistance in South Asia Using Two Wheat Recombinant Inbred Line Populations.

Spot blotch (SB) disease causes significant yield loss in wheat production in the warm and humid regions of the eastern Gangetic plains (EGP) of South Asia (SA). Most of the cultivated varieties in the eastern part of SA are affected by SB under favorable climatic conditions. To understand the nature of SB resistance and map the underlying resistant loci effective in SA, two bi-parental mapping populations were evaluated for 3 years, i.e., 2013-2015 for the BARTAI × CIANO T79 population (denoted as BC) and 2014-2016 for the CASCABEL × CIANO T79 population (CC), at Varanasi, Uttar Pradesh, India. DArTSeq genotyping-by-sequencing (GBS) platform was used for genotyping of the populations. Distribution of disease reaction of genotypes in both populations was continuous, revealing the quantitative nature of resistance. Significant "genotype," "year," and "genotype × year" interactions for SB were observed. Linkage map with the genome coverage of 8,598.3 and 9,024.7 cM in the BC and CC population, respectively, was observed. Two quantitative trait loci (QTLs) were detected on chromosomes 1A and 4D in the BC population with an average contribution of 4.01 and 12.23% of the total phenotypic variation (PV), respectively. Seven stable QTLs were detected on chromosomes 1B, 5A, 5B, 6A, 7A, and 7B in the CC population explaining 2.89-10.32% of PV and collectively 39.91% of the total PV. The QTL detected at the distal end of 5A chromosome contributed 10.32% of the total PV. The QTLs on 6A and 7B in CC could be new, and the one on 5B may represent the Sb2 gene. These QTLs could be used in SB resistance cultivar development for SA.

Metabolite profiling identified pipecolic acid as an important component of peanut seed resistance against Aspergillus flavus infection.

In a previous study, we identified a halotolerant rhizobacterium belonging to the genus Klebsiella (MBE02) that protected peanut seeds from Aspergillus flavus infection. Here, we investigated the mechanisms underlying the effect of MBE02 against A. flavus via untargeted metabolite profiling of peanut seeds treated with MBE02, A. flavus, or MBE02+A. flavus. Thirty-five metabolites were differentially accumulated across the three treatments (compared to the control), and the levels of pipecolic acid (Pip) were reduced upon A. flavus treatment only. We validated the function of Pip against A. flavus using multiple resistant and susceptible peanut cultivars. Pip accumulation was strongly associated with the resistant genotypes that also accumulated several mRNAs of the ALD1-like gene in the Pip biosynthesis pathway. Furthermore, exogenous treatment of a susceptible peanut cultivar with Pip reduced A. flavus infection in the seeds. Our findings indicate that Pip is a key component of peanut resistance to A. flavus.

Hydrogen Peroxide Prompted Lignification Affects Pathogenicity of Hemi-biotrophic Pathogen Bipolaris sorokiniana to Wheat.

Spot blotch caused by Bipolaris sorokiniana has spread to more than 9 million ha of wheat in the warm, humid areas of the Eastern Gangetic Plains (EGP) of South Asia and is a disease of major concern in other similar wheat growing regions worldwide. Differential lignin content in resistant and susceptible genotypes and its association with free radicals such as hydrogen peroxide (H2O2), superoxide (O2 -) and hydroxyl radical (OH-) were studied after inoculation under field conditions for two consecutive years. H2O2 significantly influenced lignin content in flag leaves, whereas there was a negative correlation among lignin and H2O2 to the Area Under Disease Progress Curve (AUDPC). The production of H2O2 was higher in the resistant genotypes than susceptible ones. The O2 - and OH- positively correlated with AUDPC but negatively with lignin content. This study illustrates that H2O2 has a vital role in prompting lignification and thereby resistance to spot blotch in wheat. We used cluster analysis to separate the resistant and susceptible genotypes by phenotypic and biochemical traits. H2O2 associated lignin production significantly reduced the number of appressoria and penetration pegs. We visualized the effect of lignin in disease resistance using differential histochemical staining of tissue from resistant and susceptible genotypes, which shows the variable accumulation of hydrogen peroxide and lignin around penetration sites.

High-Resolution Structural Studies Elucidate Antiatherogenic and Anti-Inflammatory Properties of Peptides Designed to Mimic Amphipathic α-Helical Domains of Apolipoprotein A-I.

Peptides designed to mimic the antiatherogenic and anti-inflammatory properties of apolipoprotein A-I show that although lipid association is required, not all lipid-associating peptides exhibit these properties. Our studies of a series of peptides showed that peptides with aromatic residues at the center of the nonpolar face were able to interact with inflammatory lipids and inhibited inflammation, which resulted in the amelioration of several lipid-mediated disorders such as lesion development, tumor formation, and Alzheimer's plaque formation. The pK a values determined using 13C nuclear magnetic resonance (NMR) spectroscopy of K residues located at the polar-nonpolar interface provided the first clue to the relative orientations of the peptide helices with respect to each other and around the edge of the lipid discoidal complexes. High-resolution 1H-NMR studies of peptide-lipid discoidal complex confirmed the amphipathic α-helical structure of the peptide, location of aromatic residues of the peptide closer to the polar-nonpolar interface, and head-to-tail arrangement of the peptide helices around the edge of the disc. Amphipathic α-helical structure and the location of aromatic residues (F, W, Y) closer to the polar-nonpolar interface in a lipid environment allow the peptide to strongly bind oxidized lipids resulting in its anti-inflammatory properties.

Natural Variation in Elicitation of Defense-Signaling Associates to Field Resistance Against the Spot Blotch Disease in Bread Wheat (Triticum aestivum L.).

Spot blotch, caused by the hemibiotropic fungus Bipolaris sorokiniana, is amongst the most damaging diseases of wheat. Still, natural variation in expression of biochemical traits that determine field resistance to spot blotch in wheat remain unaddressed. To understand how genotypic variations relate to metabolite profiles of the components of defense-signaling and the plant performance, as well as to discover novel sources of resistance against spot blotch, we have conducted field studies using 968 wheat genotypes at 5 geographical locations in South-Asia in 2 years. 46 genotypes were identified as resistant. Further, in independent confirmatory trials in subsequent 3 years, over 5 geographical locations, we re-characterized 55 genotypes for their resistance (above 46 along with Yangmai#6, a well characterized resistant genotype, and eight susceptible genotypes). We next determined time-dependent spot blotch-induced metabolite profiles of components of defense-signaling as well as levels of enzymatic components of defense pathway (such as salicylic acid (SA), phenolic acids, and redox components), and derived co-variation patterns with respect to resistance in these 55 genotypes. Spot blotch-induced SA accumulation was negatively correlated to disease progression. Amongst phenolic acids, syringic acid was most strongly inversely correlated to disease progression, indicating a defensive function, which was independently confirmed. Thus, exploring natural variation proved extremely useful in determining traits influencing phenotypic plasticity and adaptation to complex environments. Further, by overcoming environmental heterogeneity, our study identifies germplasm and biochemical traits that are deployable for spot blotch resistance in wheat along South-Asia.

Correction to: Strategic crossing of biomass and harvest index-source and sink-achieves genetic gains in wheat.

[This corrects the article DOI: 10.1007/s10681-017-2040-z.].

Frequently asked questions about chlorophyll fluorescence, the sequel.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121-158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V /F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

Growth Inhibition by External Potassium of Escherichia coli Lacking PtsN (EIIANtr) Is Caused by Potassium Limitation Mediated by YcgO.

UNLABELLED: The absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, in Escherichia coli confers a potassium-sensitive (K(s)) phenotype as the external K(+) concentration ([K(+)]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K(+) content, resulting from hyperactivated Trk-mediated K(+) uptake, is thought to cause this K(s) We provide evidence that the K(s) of the ΔptsN mutant is associated with K(+) limitation. Accordingly, the moderate K(s) displayed by the ΔptsN mutant was exacerbated in the absence of the Trk and Kup K(+) uptake transporters and was associated with reduced cellular K(+) content. Conversely, overproduction of multiple K(+) uptake proteins suppressed the K(s) Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the K(s) Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the K(s), which was correlated with elevated cellular K(+) content in the ΔptsN mutant, but the ΔptsN mutation did not alter YcgO levels. Heterologous overexpression of ycgO also led to K(s) that was associated with reduced cellular K(+) content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that K(s) in the ΔptsN mutant occurs due to K(+) limitation resulting from activation of K(+) efflux mediated by YcgO, which may be additionally stimulated by [K(+)]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity. IMPORTANCE: This study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K(+) ion metabolism in E. coli Studies on the physiological defect that renders an E. coli mutant lacking PtsN to be growth inhibited by external K(+) indicate that growth impairment results from cellular K(+) limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K(+) may stimulate K(+) limitation mediated by YcgO. It is speculated that YcgO-mediated K(+) limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.

Genomic prediction for grain zinc and iron concentrations in spring wheat.

KEY MESSAGE: Predictability estimated through cross-validation approach showed moderate to high level; hence, genomic selection approach holds great potential for biofortification breeding to enhance grain zinc and iron concentrations in wheat. Wheat (Triticum aestivum L.) is a major staple crop, providing 20 % of dietary energy and protein consumption worldwide. It is an important source of mineral micronutrients such as zinc (Zn) and iron (Fe) for resource poor consumers. Genomic selection (GS) approaches have great potential to accelerate development of Fe- and Zn-enriched wheat. Here, we present the results of large-scale genomic and phenotypic data from the HarvestPlus Association Mapping (HPAM) panel consisting of 330 diverse wheat lines to perform genomic predictions for grain Zn (GZnC) and Fe (GFeC) concentrations, thousand-kernel weight (TKW) and days to maturity (DTM) in wheat. The HPAM lines were phenotyped in three different locations in India and Mexico in two successive crop seasons (2011-12 and 2012-13) for GZnC, GFeC, TKW and DTM. The genomic prediction models revealed that the estimated prediction abilities ranged from 0.331 to 0.694 for Zn and from 0.324 to 0.734 for Fe according to different environments, whereas prediction abilities for TKW and DTM were as high as 0.76 and 0.64, respectively, suggesting that GS holds great potential in biofortification breeding to enhance grain Zn and Fe concentrations in bread wheat germplasm.

Elucidation of defense-related signaling responses to spot blotch infection in bread wheat (Triticum aestivum L.).

Spot blotch disease, caused by Bipolaris sorokiniana, is an important threat to wheat, causing an annual loss of ~17%. Under epidemic conditions, these losses may be 100%, yet the molecular responses of wheat to spot blotch remain almost uncharacterized. Moreover, defense-related phytohormone signaling genes have been poorly characterized in wheat. Here, we have identified 18 central components of salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and enhanced disease susceptibility 1 (EDS1) signaling pathways as well as the genes of the phenylpropanoid pathway in wheat. In time-course experiments, we characterized the reprogramming of expression of these pathways in two contrasting genotypes: Yangmai #6 (resistant to spot blotch) and Sonalika (susceptible to spot blotch). We further evaluated the performance of a population of recombinant inbred lines (RILs) by crossing Yangmai#6 and Sonalika (parents) and subsequent selfing to F10 under field conditions in trials at multiple locations. We characterized the reprogramming of defense-related signaling in these RILs as a consequence of spot blotch attack. During resistance to spot blotch attack, wheat strongly elicits SA signaling (SA biogenesis as well as the NPR1-dependent signaling pathway), along with WRKY33 transcription factor, followed by an enhanced expression of phenylpropanoid pathway genes. These may lead to accumulation of phenolics-based defense metabolites that may render resistance against spot blotch. JA signaling may synergistically contribute to the resistance. Failure to elicit SA (and possibly JA) signaling may lead to susceptibility against spot blotch infection in wheat.

Cationic peptide mR18L with lipid lowering properties inhibits LPS-induced systemic and liver inflammation in rats.

The cationic single domain peptide mR18L has demonstrated lipid-lowering and anti-atherogenic properties in different dyslipidemic mouse models. Lipopolysaccharide (LPS)-mediated inflammation is considered as one of the potential triggers for atherosclerosis. Here, we evaluated anti-inflammatory effects of mR18L peptide against LPS-mediated inflammation. First, we tested the efficacy and tolerance of 1, 2.5 and 5mg/kg mR18L in normolipidemic rats stimulated with 5mg/kg LPS. LPS and then mR18L were injected in different intraperitoneal regions. By 2h post LPS, mR18L inhibited LPS-mediated plasma TNF-α elevation at all doses, with the effect being stronger for 2.5mg/kg (P<0.05 vs. 1mg/kg, non-significant vs. 5mg/kg). In a similar model, 2.5mg/kg mR18L reduced LPS-mediated inflammation in the liver, as assessed by microscopic examination of liver sections and measurements of iNOS expression in the liver tissue. In plasma, 2.5mg/kg mR18L decreased levels of TNF-α and IL-6, decreased endotoxin activity and enhanced HDL binding to LPS. In another similar experiment, mR18L administered 1h post LPS, prevented elevation of plasma triglycerides by 6h post LPS and increased plasma activity of anti-oxidant enzyme paraoxonase 1, along with noted trends in reducing plasma levels of endotoxin and IL-6. Surface plasmon resonance study revealed that mR18L readily binds LPS. We conclude that mR18L exerts anti-endotoxin activity at least in part due to direct LPS-binding and LPS-neutralizing effects. We suggest that anti-endotoxin activity of mR18L is an important anti-inflammatory property, which may increase anti-atherogenic potential of this promising orally active lipid-lowering peptide.

Anti-inflammatory mechanisms of apolipoprotein A-I mimetic peptide in acute respiratory distress syndrome secondary to sepsis.

Acute respiratory distress syndrome (ARDS) due to sepsis has a high mortality rate with limited treatment options. High density lipoprotein (HDL) exerts innate protective effects in systemic inflammation. However, its role in ARDS has not been well studied. Peptides such as L-4F mimic the secondary structural features and functions of apolipoprotein (apo)A-I, the major protein component of HDL. We set out to measure changes in HDL in sepsis-mediated ARDS patients, and to study the potential of L-4F to prevent sepsis-mediated ARDS in a rodent model of lipopolysaccharide (LPS)-mediated acute lung injury, and a combination of primary human leukocytes and human ARDS serum. We also analyzed serum from non-lung disease intubated patients (controls) and sepsis-mediated ARDS patients. Compared to controls, ARDS demonstrates increased serum endotoxin and IL-6 levels, and decreased HDL, apoA-I and activity of anti-oxidant HDL-associated paraoxanase-1. L-4F inhibits the activation of isolated human leukocytes and neutrophils by ARDS serum and LPS in vitro. Further, L-4F decreased endotoxin activity and preserved anti-oxidant properties of HDL both in vitro and in vivo. In a rat model of severe endotoxemia, L-4F significantly decreased mortality and reduces lung and liver injury, even when administered 1 hour post LPS. Our study suggests the protective role of the apoA-I mimetic peptide L-4F in ARDS and gram-negative endotoxemia and warrant further clinical evaluation. The main protective mechanisms of L-4F are due to direct inhibition of endotoxin activity and preservation of HDL anti-oxidant activity.

Molecular characterization of human melanocortin-5 receptor ligand-receptor interaction.

The melanocortin-5 receptor (MC5R) is a subtype receptor of the melanocortin receptor (MCR) family, which is expressed centrally, as well as in a variety of peripheral tissues. MC5R has been implicated in many different physiological fields such as lipid metabolism and exocrine function. However, the specific molecular determinants of MC5R responsible for ligand binding and receptor signaling are currently unknown. The aim of this study is to determine the molecular basis of human MC5R (hMC5R) responsible for ligand binding and receptor signaling. Twenty-four single mutations of hMC5R were created and tested. Our results indicate that (1) substituting charged amino acid residue E92 in transmembrane domain 2 (TM2), aspartic acid 115 (D115) and D119 in TM3, and histidine (H) 257 in TM6 with alanine dramatically reduced NDP-α-MSH binding affinity and receptor signaling and (2) substituting aromatic amino acids phenylalanine (F) 195 in TM5, F254 in TM6, and H276 in TM7 with alanine also significantly decreased NDP-α-MSH binding and receptor activity. Combining pharmacological results and computer modeling, our results suggest that D115 and D119 in TM3, F195 in TM5, and F254 in TM6 may form a binding pocket for NDP-α-MSH binding. Our results provide important information about the structural aspects of hMC5R responsible for ligand binding and receptor signaling.

Lipid complex of apolipoprotein A-I mimetic peptide 4F is a novel platform for paraoxonase-1 binding and enhancing its activity and stability.

High density lipoprotein (HDL) associated paraoxonase-1 (PON1) is crucial for the anti-oxidant, anti-inflammatory, and anti-atherogenic properties of HDL. Discoidal apolipoprotein (apo)A-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) complex has been shown to be the most effective in binding PON1, stabilizing it, and enhancing its lactonase and inhibitory activity of low density lipoprotein oxidation. Based on our earlier study demonstrating that apoA-I mimetic peptide 4F forms discoidal complex with 1,2-dimyristoyl-sn-glycero-3-phosphocholine, we hypothesized that lipid complexes of 4F would be able to bind PON1 and enhance its activity and stability. To test our hypothesis, we have expressed and purified a recombinant PON1 (rPON1) and studied its interaction with 4F:POPC complex. Our studies show significant increase, compared to the control, in the paraoxonase activity and stability of rPON1 in the presence of 4F:POPC complex. We propose that 4F:POPC complex is a novel platform for PON1 binding, increasing its stability, and enhancing its enzyme activity. We propose a structural model for the 4F:POPC:PON1 ternary complex that is consistent with our results and published observations.

Two apolipoprotein E mimetic peptides with similar cholesterol reducing properties exhibit differential atheroprotective effects in LDL-R null mice.

OBJECTIVE: We investigated two apoE mimetic peptides with similar long-term plasma cholesterol reducing abilities for their effects on atherosclerotic lesions in Western diet-fed female LDL-receptor (LDL-R) null mice. METHODS AND RESULTS: Single doses of peptides Ac-hE18A-NH(2) and mR18L were administered retro-orbitally to LDL-R null mice on Western diet and plasma cholesterol was measured at 10 min, 4 h, and 24 h post administration. Peptide mR18L and not Ac-hE18A-NH(2) reduced plasma cholesterol levels significantly at 4 h post administration. However, multiple administrations (100 µg/mouse twice weekly for 8 weeks) resulted in a similar reduction in plasma cholesterol. Only the plasma from the Ac-hE18A-NH(2) group had significantly reduced reactive oxygen species levels at the end of the treatment protocol. Both mR18L and Ac-hE18A-NH(2) showed reduced atherosclerotic lesion areas. However, peptide Ac-hE18A-NH(2) was significantly more effective in inhibiting atherosclerosis. Both peptides reduced total plaque macrophage load compared to the saline treated animals, with peptide Ac-hE18A-NH(2) having a greater reduction. Incubation of HepG2 cells and THP-1 monocyte-derived macrophages with both peptides in the presence of oxidized phospholipid showed that Ac-hE18A-NH(2) promotes the secretion of apoE from the cells whereas mR18L does not. CONCLUSIONS: Despite similar reductions in plasma cholesterol levels, Ac-hE18A-NH(2) was more effective in inhibiting lesions than mR18L, possibly due to its ability to promote the secretion of apoE from hepatocytes and macrophages.

The phosphocholine-binding pocket on C-reactive protein is necessary for initial protection of mice against pneumococcal infection.

Human C-reactive protein (CRP) protects mice from lethal Streptococcus pneumoniae infection when injected into mice within the range of 6 h before to 2 h after the administration of pneumococci. Because CRP binds to phosphocholine-containing substances and subsequently activates the complement system, it has been proposed that the antipneumococcal function of CRP requires the binding of CRP to phosphocholine moieties present in pneumococcal cell wall C-polysaccharide. To test this proposal experimentally, in this study, we utilized a new CRP mutant incapable of binding to phosphocholine. Based on the structure of CRP-phosphocholine complexes, which showed that Phe(66), Thr(76), and Glu(81) formed the phosphocholine-binding pocket, we constructed a CRP mutant F66A/T76Y/E81A in which the pocket was blocked by substituting Tyr for Thr(76). When compared with wild-type CRP, mutant CRP bound more avidly to phosphoethanolamine and could be purified by affinity chromatography using phosphoethanolamine-conjugated Sepharose. Mutant CRP did not bind to phosphocholine, C-polysaccharide, or pneumococci. Mutant CRP was free in the mouse serum, and its rate of clearance in vivo was not faster than that of wild-type CRP. When either 25 µg or 150 µg of CRP was administered into mice, unlike wild-type CRP, mutant CRP did not protect mice from lethal pneumococcal infection. Mice injected with mutant CRP had higher mortality rates than mice that received wild-type CRP. Decreased survival was due to the increased bacteremia in mice treated with mutant CRP. We conclude that the phosphocholine-binding pocket on CRP is necessary for CRP-mediated initial protection of mice against lethal pneumococcal infection.

Apolipoprotein E mimetic is more effective than apolipoprotein A-I mimetic in reducing lesion formation in older female apo E null mice.

OBJECTIVE: The apolipoprotein E mimetic peptide Ac-hE18A-NH(2), capable of reducing plasma cholesterol and possessing anti-inflammatory properties, was compared with the well-studied anti-atherogenic apoA-I mimetic peptide 4F for reducing lesion formation in female apoE null mice with already existing lesions. METHODS AND RESULTS: In initial experiments, Ac-hE18A-NH(2) was administered retro-orbitally two or three times weekly for 6-8 weeks, while peptide 4F was administered intraperitoneally every day for the same period. Age matched controls were injected with saline every day. At the end of the treatment period, plasma cholesterol levels of Ac-hE18A-NH(2) administered mice were significantly lower than in 4F and control mice. However, both 4F and Ac-hE18A-NH(2) showed reduced lesion areas in en face lesion analysis to a similar extent compared to the control group, while paraoxonase-1 (PON-1) activity was increased only in the Ac-hE18A-NH(2) group. In the third experiment, both peptides were administered at the same dose, frequency, and route of administration. The reduction in en face lesions with Ac-hE18A-NH(2) was significantly greater than the 4F and control groups, although lesions in 4F-treated mice were also significantly reduced compared with controls. Both peptide groups had significantly reduced plasma lipid hydroperoxides, but only the Ac-hE18A-NH(2) group had significantly reduced serum amyloid A levels. HDL and plasma inflammatory indices were significantly reduced in both peptide groups compared with controls. CONCLUSIONS: Although both peptides had similar anti-inflammatory properties, Ac-hE18A-NH(2) was more effective in inhibiting lesions than 4F at the same dose, frequency, and route of administration, perhaps due to its cholesterol reducing properties.