Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.     

Cloning and Characterization of the Ribosomal Protein Genes in the spc Operon of a Prokaryotic Endosymbiont of the Pea Aphid, Acyrthosiphon kondoi

To correlate a prokaryotic endosymbiont in the pea aphid, Acyrthosiphonkondoi, with the endosymbionts in related aphid species as wellas with free-living bacteria and subcellular organelles, andto study the mode of its gene expression within aphid cells,we have cloned and characterized the genes encoding ribosomalproteins S3, L16, L29, S17, L14, L24, L5, S14, S8, L6, L18,S5, L30, L15 and secretion protein Y (Sec Y) from the S10 andspc ribosomal protein gene operons of this endosymbiont. Theorganization of these genes is identical to that in Escherichiacoli, and their nucleotide sequences are highly similar (87%identity) to the corresponding E. coli genes. They are muchless similar to the corresponding chloroplast and mitochondrialgenes. The guanine plus cytosine G+C content of the genes ofthe A. kondoi endosymbiont is much higher than those of theendosymbionts in related aphid species reported so far. It appearseither that the A. kondoi endosymbiont is derived from an ancestralbacterium different from those in other aphids or that its G+Ccontent increased in a relatively short time after the evolutionarydivergence of its host.     

Human CTP:phosphoethanolamine cytidylyltransferase: Enzymatic properties and unequal catalytic roles of CTP-binding motifs in two cytidylyltransferase domains

CTP:phosphoethanolamine cytidylyltransferase (ECT) is a key enzyme in the CDP-ethanolamine branch of the Kennedy pathway, which is the primary pathway of phosphatidylethanolamine (PE) synthesis in mammalian cells. Here, the enzymatic properties of recombinant human ECT (hECT) were characterized. The catalytic reaction of hECT obeyed Michaelis–Menten kinetics with respect to both CTP and phosphoethanolamine. hECT is composed of two tandem cytidylyltransferase (CT) domains as ECTs of other organisms. The histidines, especially the first histidine, in the CTP-binding motif HxGH in the N-terminal CT domain were critical for its catalytic activity in vitro, while those in the C-terminal CT domain were not. Overexpression of the wild-type hECT and hECT mutants containing amino acid substitutions in the HxGH motif in the C-terminal CT domain suppressed the growth defect of the Saccharomyces cerevisiae mutant of ECT1 encoding ECT in the absence of a PE supply via the decarboxylation of phosphatidylserine, but overexpression of hECT mutants of the N-terminal CT domain did not. These results suggest that the N-terminal CT domain of hECT contributes to its catalytic reaction, but C-terminal CT domain does not.     

Stimulation of Expression of a Silica-Induced Protein (Sip) in Thermus thermophilus by Supersaturated Silicic Acid

The effects of silicic acid on the growth of Thermus thermophilus TMY, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in Japan, were examined at 75°C. At concentrations higher than the solubility of amorphous silica (400 to 700 ppm SiO₂), a silica-induced protein (Sip) was isolated from the cell envelope fraction of log-phase TMY cells grown in the presence of supersaturated silicic acid. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the molecular mass and pI of Sip to be about 35 kDa and 9.5, respectively. Induction of Sip expression occurred within 1 h after the addition of a supersaturating concentration of silicic acid to TM broth. Expression of Sip-like proteins was also observed in other thermophiles, including T. thermophilus HB8 and Thermus aquaticus YT-1. The amino acid sequence of Sip was similar to that of the predicted solute-binding protein of the Fe³⁺ ABC transporter in T. thermophilus HB8 (locus tag, TTHA1628; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_006461","term_id":"55979969","term_text":"NC_006461"}}NC_006461; GeneID, 3169376). The sip gene (987-bp) product showed 87% identity with the TTHA1628 product and the presumed Fe³⁺-binding protein of T. thermophilus HB27 (locus tag TTC1264; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_005835","term_id":"46198308","term_text":"NC_005835"}}NC_005835; GeneID, 2774619). Within the genome, sip is situated as a component of the Fbp-type ABC transporter operon, which contains a palindromic structure immediately downstream of sip. This structure is conserved in other T. thermophilus genomes and may function as a terminator that causes definitive Sip expression in response to silica stress.Occurring mainly in the form of silica (SiO₂), silicon (Si) is the second-most abundant element in the earth''s crust, accounting for 28.8% of the earth''s mass (34). SiO₂ exists as monosilicic acid (Si(OH)₄) in aqueous solution, as represented in the following equation: SiO₂ + 2H₂O · (Si(OH)₄). The solubility of silica greatly depends on temperature, pH (17), and salt concentration, among other parameters (27). As the temperature of a silicic acid solution declines, its concentration can exceed the solubility of amorphous silica. Under those conditions, silicic acid polymerizes to form polysilisic acid, which is relatively stable in aqueous solution because the repulsion between the negative charges on its surface keeps it from readily aggregating and precipitating. In a geothermal reservoir, at high temperature and pressure, the silicic acid concentration at equilibrium shows the solubility of quartz. However, when that geothermal water is discharged to the surface, the silicic acid concentration becomes supersaturated as the water boils, frequently leading to the formation of siliceous deposits called “silica sinter” (11). Microscopic observation of such siliceous deposits reveals many microbe-like structures (20), and it has been suggested that these fossils represent archean microorganisms that grew in the hot, supersaturated fluids (26). There have been a number of experimental studies carried out with the aim of characterizing the physical changes associated with various bacteria during silicification (23, 26, 31, 33, 37); however, the effect of silica on the bacterial habitat in geothermal environments and the mechanism by which siliceous deposits are formed remain unexplained.Recent studies have shown that biosilicification in geothermal areas reflects the activities of various thermophilic microorganisms (15, 23, 29). For instance, geothermal water and the water discharged from hydrothermal vents contain a high concentration of silicic acid, and biogenic textures covered with amorphous silica have been found in areas around both sources (12, 22). Moreover, in a study of experimental silicification, interactions were observed between silica and Sulfurihydrogenibium azorense, a representative member of the order Aquificales (26). Still, the effect of silica on the bacterial habitat in thermal environments and the molecular properties affecting aggregation and siliceous deposition remain poorly understood.Our previous studies have focused on the effect of bacteria on the formation of siliceous deposits in geothermal water (18, 21). Siliceous deposits (called silica scale) that form in pipelines and on surface equipment in geothermal power plants cause serious economic problems related to energy loss and to plant maintenance throughout the world (38). We also observed that a Thermus strain isolated from silica scale (TMY) was able to efficiently generate and deposit amorphous silica in vitro, beginning in the latter part of the log growth phase (19). On the basis of its morphological, physiological, and genetic properties, the organism was identified as a strain of Thermus thermophilus, and its distinct properties were indicative of the microdiversity of T. thermophilus strains (14). Thermus strains are the predominant heterotrophs in natural geothermal and hydrothermal habitats, and thus far, the genomes of T. thermophilus strains HB8 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AP008226","term_id":"55771382","term_text":"AP008226"}}AP008226) and HB27 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE017221","term_id":"46197919","term_text":"AE017221"}}AE017221) have been sequenced completely (16). Given its genomic similarity to T. thermophilus HB8, we analyzed the genetic information of strain TMY in that context.Here we report the effect of silicic acid concentration on the growth of T. thermophilus TMY, which was isolated from a siliceous deposit formed at a geothermal electric power plant. Notably, supersaturated silicic acid markedly stimulated expression of one cell envelope protein, which we named silica-induced protein (Sip). Induction of Sip expression occurred rapidly after the cells were exposed to supersaturated silicic acid, and the amino acid sequence of Sip showed significant similarity with the Fe³⁺ ABC transporters observed in other Thermus strains. These results shed new light on the growth and biosilisification associated with thermophiles in geothermal environments.     

A case of giardiasis expressing severe systemic symptoms and marked hypereosinophilia

An 88-year-old Japanese woman was referred to our hospital due to a one-month history of face edema, aphagia, shortness of breath, and skin rush over almost her entire skin. She had no abdominal symptoms. Her peripheral blood count showed a white blood cell (WBC) count of 27.1 × 10⁹/L with 82.1% eosinophils. Serum non-specific Immunoglobulin E was within a normal range. Soluble interleukin-2 receptor was elevated to 4200 U/mL. At first, her eosinophil count was so high that we suspected she had an eosinophilic leukemia or hypereosinophilic syndrome. After admission, cysts of Giardia duodenalis (G. duodenalis) were detected in the patient's feces by microscopic analysis, then she was diagnosed with giardiasis, and 750 mg per day of metronidazole was administered for seven days. Her WBC count decreased to 6.0 × 10⁹/L with 10% eosinophils, and her systemic symptoms improved. At that time her serum IL-5 was within a normal range. A few months later, the patient again complained of skin rush, and G. duodenalis was once again found in her feces. Her serum IL-5 was elevated to 751 pg/mL. Metronidazole was administered for two weeks, and her eosinophil count decreased. G. duodenalis is a protozoan parasite, and it is one of the most common waterborne transmission gastrointestinal parasites in the world. G. duodenalis rarely causes hypereosinophilia. To our knowledge, this is the first case report of giardiasis with extreme hypereosinophilia and severe systemic symptoms.     

Pigment shuffling in antenna systems achieved by expressing prokaryotic chlorophyllide a oxygenase in Arabidopsis

The organization of pigment molecules in photosystems is strictly determined. The peripheral antennae have both chlorophyll a and b, but the core antennae consist of only chlorophyll a in green plants. Furthermore, according to the recent model obtained from the crystal structure of light-harvesting chlorophyll a/b-protein complexes II (LHCII), individual chlorophyll-binding sites are occupied by either chlorophyll a or chlorophyll b. In this study, we succeeded in altering these pigment organizations by introducing a prokaryotic chlorophyll b synthesis gene (chlorophyllide a oxygenase (CAO)) into Arabidopsis. In these transgenic plants (Prochlirothrix hollandica CAO plants), approximately 40% of chlorophyll a of the core antenna complexes was replaced by chlorophyll b in both photosystems. Chlorophyll a/b ratios of LHCII also decreased from 1.3 to 0.8 in PhCAO plants. Surprisingly, these transgenic plants were capable of photosynthetic growth similar to wild type under low light conditions. These results indicate that chlorophyll organizations are not solely determined by the binding affinities, but they are also controlled by CAO. These data also suggest that strict organizations of chlorophyll molecules are not essential for photosynthesis under low light conditions.     

Involvement of AtNAP1 in the regulation of chlorophyll degradation in Arabidopsis thaliana

In plants, chlorophyll is actively synthesized from glutamate in the developmental phase and is degraded into non-fluorescent chlorophyll catabolites during senescence. The chlorophyll metabolism must be strictly regulated because chlorophylls and their intermediate molecules generate reactive oxygen species. Many mechanisms have been proposed for the regulation of chlorophyll synthesis including gene expression, protein stability, and feedback inhibition. However, information on the regulation of chlorophyll degradation is limited. The conversion of chlorophyll b to chlorophyll a is the first step of chlorophyll degradation. In order to understand the regulatory mechanism of this reaction, we isolated a mutant which accumulates 7-hydroxymethyl chlorophyll a (HMChl), an intermediate molecule of chlorophyll b to chlorophyll a conversion, and designated the mutant hmc1. In addition to HMChl, hmc1 accumulated pheophorbide a, a chlorophyll degradation product, when chlorophyll degradation was induced by dark incubation. These results indicate that the activities of HMChl reductase (HAR) and pheophorbide a oxygenase (PaO) are simultaneously down-regulated in this mutant. We identified a mutation in the AtNAP1 gene, which encodes a subunit of the complex for iron–sulfur cluster formation. HAR and PaO use ferredoxin as a reducing power and PaO has an iron-sulfur center; however, there were no distinct differences in the protein levels of ferredoxin and PaO between wild type and hmc1. The concerted regulation of chlorophyll degradation is discussed in relation to the function of AtNAP1.     

Effects of retinol binding protein-4 on vascular endothelial cells

The study was designed to investigate the effect of retinol binding protein (RBP)-4 on the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, which mediate the effects of insulin in vascular endothelial cells. The effects of RBP4 on nitric oxide (NO) and insulin-stimulated endothelin-1 (ET-1) secretion and on phosphorylation (p) of Akt, endothelial NO synthetase (eNOS), and extracellular signal-regulated kinase (ERK)1/2 were investigated in bovine vascular aortic endothelial cells (BAECs). RBP4 showed an acute vasodilatatory effect on aortic rings of rats within a few minutes. In BAECs, RBP4-treatment for 5 min significantly increased NO production, but inhibited insulin-stimulated ET-1 secretion. RBP4-induced NO production was not inhibited by tetraacetoxymethylester (BAPTA-AM), an intracellular calcium chelator, but was completely abolished by wortmannin, a PI3K inhibitor. RBP4 significantly increased p-Akt and p-eNOS production, and significantly inhibited p-ERK1/2 production. Triciribine, an Akt inhibitor, and wortmannin significantly inhibited RBP4-induced p-Akt and p-eNOS production. Inhibition of Akt1 by small interfering RNA decreased p-eNOS production enhanced by RBP4 in human umbilical vein endothelial cells. In conclusion, RBP4 has a robust acute effect of enhancement of NO production via stimulation of part of the PI3K/Akt/eNOS pathway and inhibition of ERK1/2 phosphorylation and insulin-induced ET-1 secretion, probably in the MAPK pathway, which results in vasodilatation.     

Photochemical energy conversion system using immobilized chloroplasts

Immobilized chloroplasts and Clostridium butyricum were employed for a photochemical energy conversion system. Spinach chloroplasts were immobilized in 2% agar gel. The optimum temperature of immobilized chloroplasts was 30°C. The maximum activity was obtained in the phosphate buffer solution (pH 8.0) containing 8μM of ferredoxin under an N₂ bubbling condition. Hydrogen was evolved under illumination by immobilized chloroplasts and C. butyricum. Hydrogen produced by this system was applied to a hydrogen-oxygen fuel cell. Photoinduced current was obtained from this photochemical energy conversion system. A photocurrent of 0.4?1.5 mA was continuously obtained for 4 h. The conversion ratio from hydrogen to current was 80?100%.     

Identification of the RelA domain responsible for action of a new NF-kappaB inhibitor DHMEQ

A new NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) has a potential to be applied to clinical medicine as an anti-cancer and anti-inflammatory agent. DHMEQ inhibits localization of NF-κB in the nucleus and the inhibitory effect by DHMEQ is more potent on p50/RelA than on p50 homodimer. However, a molecular target of DHMEQ is unknown. In this study, we identified residues CEGRSAGSI, which appear in RelA (amino acids 38-46), c-Rel (28-36), and RelB (144-152), but not in p50 and p52, as a target of DHMEQ. As a possible mechanism, we propose that DHMEQ accesses CEGRSAGSI domain recognizing RSAGSI structure and directly binds to cysteine. This target domain appears to be unique among mammalian proteins. The results obtained in this study may provide better understanding of the action of DHMEQ and a key for developing a new NF-κB inhibitor with more potent activity.