Systematic phenome analysis of Escherichia coli multiple‐knockout mutants reveals hidden reactions in central carbon metabolism

Central carbon metabolism is a basic and exhaustively analyzed pathway. However, the intrinsic robustness of the pathway might still conceal uncharacterized reactions. To test this hypothesis, we constructed systematic multiple‐knockout mutants involved in central carbon catabolism in Escherichia coli and tested their growth under 12 different nutrient conditions. Differences between in silico predictions and experimental growth indicated that unreported reactions existed within this extensively analyzed metabolic network. These putative reactions were then confirmed by metabolome analysis and in vitro enzymatic assays. Novel reactions regarding the breakdown of sedoheptulose‐7‐phosphate to erythrose‐4‐phosphate and dihydroxyacetone phosphate were observed in transaldolase‐deficient mutants, without any noticeable changes in gene expression. These reactions, triggered by an accumulation of sedoheptulose‐7‐phosphate, were catalyzed by the universally conserved glycolytic enzymes ATP‐dependent phosphofructokinase and aldolase. The emergence of an alternative pathway not requiring any changes in gene expression, but rather relying on the accumulation of an intermediate metabolite may be a novel mechanism mediating the robustness of these metabolic networks.     

Protection against malaria due to innate immunity enhanced by low-protein diet

Mice were fed ad libitum with a normal diet (25% protein) or low-protein diets (0-12.5% protein) for a wk and then infected with a nonlethal or lethal strain of Plasmodium yoelii, that is, blood stage infection. The same diet was continued until recovery. Mice fed with a normal diet showed severe parasitemia during nonlethal infection, but survived the infection. They died within 2 wk in the case of lethal infection. However, all mice fed with low-protein diets survived without apparent parasitemia (there were small peaks of parasitemia) in cases of both nonlethal and lethal strains. These surviving mice were found to have acquired potent innate immunity, showing the expansion of NK1.1 -TCRint cells and the production of autoantibodies during malarial infection. Severe combined immunodeficiency (scid) mice, which lack TCRint cells as well as TCRhigh cells, did not survive after malarial infection of lethal strain of P. yoelii, even when low-protein diets were given. These results suggest that low-protein diets enhanced innate immunity and inversely decreased conventional immunity, and that these immunological deviations rendered mice resistant against malaria. The present outcome also reminds us of our experience in the field study of malaria, in which some inhabitants eventually avoided contracting malaria even after apparent malarial infection.     

Pfcrt genotypes and related microsatellite DNA polymorphisms on Plasmodium falciparum differed among populations in the Greater Mekong Subregion

Malaria morbidity and mortality have decreased gradually in the Greater Mekong Subregion (GMS). Presently, WHO sets a goal to eliminate malaria by 2030 in the GMS. However, drug-resistant malaria has been reported from several endemic areas. To achieve the goal of elimination, the status of the emergence and spread of drug resistance should be monitored. In this study, the genotype of the Plasmodium falciparum chloroquine (CQ) resistance transporter gene (pfcrt) and 6 microsatellite DNA loci flanking the gene were examined. P. falciparum isolates (n?=?136) was collected from malaria patients in Thailand (n?=?50, 2002–2005), Vietnam (n?=?39, 2004), Laos (n?=?15, 2007) and Cambodia (n?=?32, 2009). Amino acid sequences at codons 72–76 on the gene were determined. All of the isolates from Thailand were CQ-resistant (CVIET), as were all of the isolates from Cambodia (CVIET, CVIDT). Thirteen of the 15 isolates (87%) from Laos were CQ-resistant (CVIET, CVIDT), whereas the other 2 (13%) were CQ-susceptible (CVMNK). In contrast, 27 of the 39 isolates (69%) from Vietnam were CQ-susceptible (CVMNK), whereas the other 12 (31%) were CQ-resistant (CVIET, CVIDT, CVMDT) or mixed (CVMNK/CVIDT). The mean of expected heterozygosity of the microsatellite loci was 0.444 in the Thai population, 0.482 in the Cambodian population, and 0.734 in the Vietnamese population. Genetic diversity in the Thai population was significantly lower than that in the Vietnamese population. These results suggested that chloroquine selective pressure on P. falciparum populations is heterogeneous in the GMS. Therefore, further examination to understand the mechanisms behind the emergence and spread of drug-resistant malaria are needed.     

Selective activation of extrathymic T cells in the liver by glycyrrhizin

Extrathymic pathways for T cell differentiation were recently demonstrated in the liver, intestine and omentum. In this study, glycyrrhizin (GL), a plant extract was investigated as to its effect on extrathymic T cells in the liver of mice. A new method using anti-LFA-1 mAb in conjunction with anti-TCR or -CD3 mAbs to sensitively identify such extrathymic T cells is included. Single injection and repeated injections of GL increased not only the number of total hepatic MNC but also the proportion of intermediate TCR cells, which are extrathymic T cells uniquely seen in the liver. In contrast to other tested reagents (e.g., lymphotoxin and estrogen) that activated the extrathymic T cells and simultaneously induced profound thymic atrophy, GL did not affect regular T cells in the thymus. The present results suggest that the selective activation of extrathymic T cells in the liver might be intimately related to the clinical effects of GL.     

Systematic characterization of curved DNA segments randomly cloned from Escherichia coli and their functional significance

Summary In addition to the set of curved DNA segments isolated previously from Escherichia coli, another set of curved DNA segments has now been isolated. To gain an insight into the functional significance of these curved DNA sequences, systematic analyses were carried out, which included not only mapping of the precise locations of the segments on the E. coli chromosome but also clarification of the gene organization in the chromosomal regions surrounding the curved DNA sequences. It was demonstrated that most of the curved DNA sequences, which have been characterized so far, appear to be located immediately upstream of the coding sequences of adjacent genes. It was also demonstrated that an E. coli histone-like protein, named H-NS (or H1a), exhibits a strong affinity for naturally occurring curved DNA sequences in regions upstream promoters.     

Multiple ligand-binding properties of the lipocalin member chicken alpha1-acid glycoprotein studied by circular dichroism and electronic absorption spectroscopy: the essential role of the conserved tryptophan residue

Multiple ligand-binding properties of the 30-kDa chicken alpha(1)-acid glycoprotein (cAGP), a member of the lipocalin protein family, were investigated for the first time by using circular dichroism (CD) and UV/Vis absorption spectroscopy methods. By measuring induced CD (ICD) spectra, high-affinity binding (K(a) approximately 10(5)-10(6) M(-1)) of several drugs, dyes and natural compounds to cAGP was demonstrated including antimalarial agents (quinacrine, primaquine), phenotiazines (chlorpromazine, methylene blue), propranolol, non-steroidal antiinflammatory drugs (ketoprofen, diclofenac), tamoxifen, diazepam, tacrine, dicoumarol, cationic dyes (auramine O, thioflavine T, ethidium bromide), benzo[a]pyrene, L-thyroxine, bile pigments (bilirubin, biliverdin), alkaloids (piperine, aristolochic acid), saturated and unsaturated fatty acids. Analysis of the extrinsic CD spectra with the study of the covalently modified protein and CD displacement experiments revealed that a single Trp26 residue of cAGP conserved in the whole lipocalin family is part of the binding site, and it is essentially involved in the ligand-binding process via pi-pi stacking interaction resulting in the appearance of strong induced CD bands due to the non-degenerate intermolecular exciton coupling between the pi-pi* transitions of the stacked indole ring-ligand chromophore. The finding that cAGP is able to accommodate a broad spectrum of ligands belonging to different chemical classes suggests that its core beta-barrel cavity is unusually wide containing overlapping sub-sites. Significance of these new data in understanding of the ligand-binding properties of other lipocalins, especially that of human AGP, and potential practical applications are briefly discussed. Overall, cAGP serves as a simple, ultimate model to extend our knowledge on ligand-binding properties of lipocalins and to study the role of tryptophan residues in molecular recognition processes.     

Effect of randomly interesterified triacylglycerol containing medium- and long-chain fatty acids on hepatic fatty acid oxidation after a single administration to rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.     

Radioresistance of Intermediate TCR Cells and Their Localization in the Body of Mice Revealed by Irradiation

Extrathymic generation of T cells in the liver and in the intestine was recently demonstrated. We investigated herein whether such T cells, especially those in the liver, are present in other organs of mice. This investigation is possible employing our recently introduced method with which even a minor proportion of extrathymic, intermediate TCR cells in organs other than the liver can be identified. Intermediate TCR cells expressed higher levels of IL-2Rβ and LFA-1 than bright TCR cells (i.e., T cells of thymic origin) as revealed by two-color staining. Although intermediate TCR cells were present at a small proportion in the spleen and thymus, they predominated in these organs after irradiation (9 Gy) and bone marrow reconstitution, or after low dose irradiation (6 Gy). This was due to that intermediate TCR cells were relatively radioresistant, whereas bright TCR cells were radiosensitive. Microscopic observation and immunochemical staining showed that intermediate TCR cells in the spleen localized in the red pulp and those in the thymus localized in the medulla. These intermediate TCR cells displayed a large light scatter, similar to such cells in the liver. The present results suggest that intermediate TCR cells may proliferate at multiple sites in the body.     

Protection against malaria by anti-erythropoietin antibody due to suppression of erythropoiesis in the liver and at other sites

We have previously reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection. At this time, erythropoietin (EPO) is elevated in the serum. In the present study, we administered EPO or anti-EPO antibody into C57BL/6 (B6) mice to modulate the serum level of EPO. When mice were infected with a non-lethal strain (17NXL) of Plasmodium yoelii (blood-stage infection of 10(4) parasitized erythrocytes per mouse), parasitemia continued for 1 month, showing a peak at day 17. Daily injection of EPO (200 IU/day per mouse) from day five to day 14 prolonged parasitemia, whereas injection of anti-EPO antibody (1.5 mg/day per mouse) every second day from day five to day 28 decreased it. Erythropoiesis was confirmed in the liver, spleen and bone marrow by the appearance of nucleated erythrocytes (TER119+). When anti-EPO antibody was injected by the same protocol into mice infected with a lethal strain (17XL) of P. yoelii, all mice showed decreased parasitemia and recovered from the infection. These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria.