Glutamine cycling in isolated working rat heart

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.     

Diverse peptide presentation of rhesus macaque major histocompatibility complex class I Mamu-A 02 revealed by two peptide complex structures and insights into immune escape of simian immunodeficiency virus

Major histocompatibility complex class I (MHC I)-restricted CD8(+) T-cell responses play a pivotal role in anti-human immunodeficiency virus (HIV) immunity and the control of viremia. The rhesus macaque is an important animal model for HIV-related research. Among the MHC I alleles of the rhesus macaque, Mamu-A 02 is prevalent, presenting in ≥20% of macaques. In this study, we determined the crystal structure of Mamu-A 02, the second structure-determined MHC I from the rhesus macaque after Mamu-A 01. The peptide presentation characteristics of Mamu-A 02 are exhibited in complex structures with two typical Mamu-A 02-restricted CD8(+) T-cell epitopes, YY9 (Nef159 to -167; YTSGPGIRY) and GY9 (Gag71 to -79; GSENLKSLY), derived from simian immunodeficiency virus (SIV). These two peptides utilize similar primary anchor residues (Ser or Thr) at position 2 and Tyr at position 9. However, the central region of YY9 is different from that of GY9, a difference that may correlate with the immunogenic variance of these peptides. Further analysis indicated that the distinct conformations of these two peptides are modulated by four flexible residues in the Mamu-A 02 peptide-binding groove. The rare combination of these four residues in Mamu-A 02 leads to a variant presentation for peptides with different residues in their central regions. Additionally, in the two structures of the Mamu-A 02 complex, we compared the binding of rhesus and human β(2) microglobulin (β(2)m) to Mamu-A 02. We found that the peptide presentation of Mamu-A 02 is not affected by the interspecies interaction with human β(2)m. Our work broadens the understanding of CD8(+) T-cell-specific immunity against SIV in the rhesus macaque.     

The <Emphasis Type="Italic">luxS</Emphasis> Gene Is Involved in AI-2 Production,Pathogenicity, and Some Phenotypes in <Emphasis Type="Italic">Erwinia amylovora</Emphasis>

Erwinia amylovora causes fire blight of apple, pear, and other members of the Rosaceae family. The enzyme LuxS catalyzes the last step in the production of autoinducer-2 (AI-2), a molecule implicated with quorum sensing in many bacterial species. It is now well recognized that LuxS also plays a central role in sulfur metabolism and in the activated methyl cycle, which is responsible for the generation of S-adenosyl-l-methionine. A research paper has reported that luxS is not involved with quorum sensing in Er. amylovora, but in our study, Er. amylovora strain NCPPB1665 (Ea1665) produced luxS-dependent extracellular AI-2 activity. Additionally, the maximal AI-2 activity occurred during late-exponential and early-stationary growth phases and diminished during the stationary phase. The luxS mutant of Ea1665 was constructed, and the phenotypes of a defined luxS mutant have been characterized. Inactivation of luxS in Ea1665 impaired motility, extracellular polysaccharide (EPS) production, and tolerance for hydrogen peroxide, and reduced virulence on pear leaves. Yan Gao and Junxian Song contributed equally to this research.     

Antibody response to heat shock proteins and histopathology in mice infected with Trypanosoma cruzi and maintained at elevated temperature

Highly susceptible C3HeB/FeJ mice survive an otherwise lethal infection with a Brazil strain of Trypanosoma cruzi when held at an elevated environmental temperature of 36 C. The body temperature of these mice has been shown to increase 3-4 C to levels typical of a febrile response. In the present study, the synthesis of parasite heat shock proteins (hsp60, hsp70, and hsp90) was shown to be enhanced at a temperature of 39 C and the results of immunoprecipitation analysis indicated that parasite HSPs are highly immunogenic in T. cruzi-infected mice maintained at 36 C or room temperature (RT). Differences in the histopathology of cardiac and skeletal muscle in C3HeB/FeJ mice maintained at RT or 36 C at different times postinfection also were investigated in this study. The lower numbers of circulating parasites observed in mice maintained at 36 C were correlated with lower levels of tissue parasitism, inflammation, and tissue destruction. Finally, the transfer of infected mice from RT to an environment of 36 C at various times during infection was shown to increase the survival rate of infected mice and also resulted in a dramatic reduction in parasitemia levels. In light of the growing evidence for a beneficial effect of elevated temperature during experimental Chagas' disease, further studies seem warranted to determine if hyperthermia or fever therapy might also be beneficial in the treatment of humans infected with T. cruzi.     

Analysis of DNA sequences of six chloroplast and nuclear genes suggests incongruence, introgression, and incomplete lineage sorting in the evolution of Lespedeza (Fabaceae)

The genus Lespedeza (Fabaceae) consists of 40 species disjunctively distributed in East Asia and eastern North America. Phylogenetic relationships of all Lespedeza species and closely related genera were reconstructed using maximum parsimony, maximum likelihood, and Bayesian analyses of sequence data from five chloroplast (rpl16, rpl32-trnL, rps16-trnQ, trnL-F, and trnK/matK) and one nuclear (ITS) DNA regions. All analyses yielded consistent relationships among major lineages. Our results suggested that Campylotropis, Kummerowia, and Lespedeza are monophyletic, respectively. Lespedeza is resolved as sister to Kummerowia and these two together are further sister to Campylotropis. Neither of the two subgenera, subgen. Lespedeza and subgen. Macrolespedeza, in Lespedeza based on morphological characters, is recovered as monophyletic. Within Lespedeza, the North American clade is retrieved as sister to the Asian clade. The nuclear and chloroplast markers showed incongruent phylogenetic signals at shallow-level phylogeny, which may point to either introgression or incomplete lineage sorting in Lespedeza. The divergence times within Lespedeza and among related genera were estimated using Bayesian approach with BEAST. It is assumed that following the divergence between Kummerowia and Lespedeza in Asia in the late Miocene, the ancestor of Lespedeza diverged into the North American and the Asian lineages. The North American ancestor quickly migrated to North America through the Bering land bridge in the late Miocene. The North American and Asian lineages started to diversify almost simultaneously in the late Miocene but resulted in biased numbers of species in two continents.     

The roles of veratryl alcohol and nonionic surfactant in the oxidation of phenolic compounds by lignin peroxidase

Veratryl alcohol (VA) at higher concentration stimulated the lignin peroxidase (LiP)-catalyzed oxidation of phenolic compounds remarkably. This novel phenomenon was due to its competition with the phenols for the active site of the enzyme and to the high reactivity of the formed cation radical of VA (VA+*) which resulted in an additional oxidation of the phenols. The influence of the nonionic surfactant Tween 80 on the VA-enhanced LiP-catalyzed oxidation of phenols depended on its concentration. At lower concentration it had a small synergetic effect but at higher concentration it decreased the initial rate. Studies of the capillary electrophoretic behavior of LiP in the presence of Tween 80 showed that this effect was caused by the surfactant aggregation on LiP which, at higher surfactant concentrations, might impede the access of VA to its binding site on LiP and, consequently, the VA+* formation.     

Hepatitis B virus surface antigen assembly function persists when entire transmembrane domains 1 and 3 are replaced by a heterologous transmembrane sequence

Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.     

Formation of soil organic carbon pool is regulated by the structure of dissolved organic matter and microbial carbon pump efficacy: A decadal study comparing different carbon management strategies

To achieve long-term increases in soil organic carbon (SOC) storage, it is essential to understand the effects of carbon management strategies on SOC formation pathways, particularly through changes in microbial necromass carbon (MNC) and dissolved organic carbon (DOC). Using a 14-year field study, we demonstrate that both biochar and maize straw lifted the SOC ceiling, but through different pathways. Biochar, while raising SOC and DOC content, decreased substrate degradability by increasing carbon aromaticity. This resulted in suppressed microbial abundance and enzyme activity, which lowered soil respiration, weakened in vivo turnover and ex vivo modification for MNC production (i.e., low microbial carbon pump “efficacy”), and led to lower efficiency in decomposing MNC, ultimately resulting in the net accumulation of SOC and MNC. In contrast, straw incorporation increased the content and decreased the aromaticity of SOC and DOC. The enhanced SOC degradability and soil nutrient content, such as total nitrogen and total phosphorous, stimulated the microbial population and activity, thereby boosting soil respiration and enhancing microbial carbon pump “efficacy” for MNC production. The total C added to biochar and straw plots were estimated as 27.3–54.5 and 41.4 Mg C ha⁻¹, respectively. Our results demonstrated that biochar was more efficient in lifting the SOC stock via exogenous stable carbon input and MNC stabilization, although the latter showed low “efficacy”. Meanwhile, straw incorporation significantly promoted net MNC accumulation but also stimulated SOC mineralization, resulting in a smaller increase in SOC content (by 50%) compared to biochar (by 53%–102%). The results address the decadal-scale effects of biochar and straw application on the formation of the stable organic carbon pool in soil, and understanding the causal mechanisms can allow field practices to maximize SOC content.     

Construction of a recombinant BHV-1 expressing the VP1 gene of foot and mouth disease virus and its immunogenicity in a rabbit model

Foot-and-mouth disease (FMD) and infectious bovine rhinotracheitis (IBR) are two important infectious diseases of cattle. Using bovine herpesvirus type 1 (BHV-1) as a gene delivery vector for development of live-viral vaccines has gained widespread interest. In this study, a recombinant BHV-1 was constructed by inserting the synthetic FMDV (O/China/99) VP1 gene in the the gE locus of BHV-1 genome under the control of immediately early gene promoter of human cytomegalovirus (phIE CMV) and bovine growth hormone polyadenylation (BGH polyA) signal. After homologous recombination and plaque purification, a recombinant virus named BHV-1/gE⁻/VP1 was acquired and identified. The immunogenicity was confirmed in a rabbit model by virus neutralization test and enzyme-linked immunosorbent assay (ELISA). The result indicated that the BHV-1/gE⁻/VP1 has the potential for being developed as a bivalent vaccine for FMD and IBR.