Isolation of a cAMP-requiring mutant in Escherichia coli K-12: evidence of growth regulation via N-acetylglucosamine metabolism controlled by cAMP

Abstract An adenylate cyclase gene ( cya ) mutant was mutagenized and an adenosine 3,5-cyclic monophosphate (cAMP)-requiring mutant (KM8161) was obtained on Davis minimal medium containing glucose in the presence or absence of cAMP. KM8161 also required N -acetylglucosamine for its growth instead of cAMP. Furthermore, the mutant could use neither glucosamine nor N -acetylglucosamine as the carbon source. These results indicate that the cAMP-requiring property is due to multiple mutations of a few genes involved in amino sugar metabolism in addition to cya . By genetic analysis of KM8161, one gene, which was tentatively termed cidA and located near 2 min on the chromosomal map, proved to be defective. Reversion of cidA mutation in KM8161 resulted in recovery of not only the cAMP-requiring phenotype but also non-utilization of amino sugars. When both cAMP and N -acetylglucosamine or glucosamine were added to the culture medium for KM8161, only N -acetylglucosamine could be utilized as the carbon source. These studies s strongly suggest that the cidA or cya mutation in KM8161 causes deficiency in different stages of amino sugar metabolism and the regulatory circuit of growth by cAMP is mediated via control of N -acetylglucosamine metabolism.     

Intranuclear microinjection for transformation of tomato callus cells

An efficient method, called the culture plate method, was devised for microinjection of foreign materials into nuclei of tomato callus cells. The culture plate method, used in this study, is advantageous because cells suitable for microinjection can be selected microscopically and the injected cells subsequently cultured in the same plate. With this microinjection system, some foreign materials were injected into nuclei of callus cells without causing detrimental effects. Kanamycin-resistant callus clones were obtained 1 month after injection from single cells whose nuclei were microinjected with a NPT II DNA fragment of the pE2KX plasmid.     

A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.     

Development of simple sequence repeat markers and construction of a high-density linkage map of Capsicum annuum

To facilitate marker-assisted breeding and genetic analyses of pepper (Capsicum annuum), we developed non-redundant 2- or 3-base simple sequence repeat (SSR) markers from enriched C. annuum genomic libraries and from C. annuum cDNA sequences in public databases. The SSR-enriched libraries were constructed using combinations of three restriction enzymes (AluI, HaeIII, and RsaI) and two biotinylated oligonucleotides [b(GA)₁₅ and b(CA)₁₅]. Ultimately, we obtained 1,736 genomic SSR markers and 1,344 cDNA-derived SSR markers from 6,528 clones and 13,003 sequences, respectively. We mapped 597 markers, including 265 of the newly developed SSR markers, onto a linkage map by using doubled-haploid (DH) lines derived from an intraspecific cross of two pure lines of C. annuum (K9-11 × MZC-180). The map, designated as the KL-DH map, consisted of 12 linkage groups. The map covered a genetic distance of 2,028 cM, and the average distance between markers was less than 4 cM. The frame structure of the KL-DH map was compared with the published standard conserved ortholog set II (COSII) map, which was derived from an interspecific F₂ population (C. frutescens × C. annuum), by using tomato (Solanum lycopersicum) chromosomal sequences to bridge the two maps. The intraspecific KL-DH map constructed in this study and the interspecific COSII map were similar in map length and marker distribution, suggesting that the KL-DH map covers nearly the whole genome of C. annuum.     

Rates of Serious Intracellular Infections in Autoimmune Disease Patients Receiving Initial Glucocorticoid Therapy

Background/Aims

The Japanese National Hospital Organization evidence-based medicine (EBM) Study group for Adverse effects of Corticosteroid therapy (J-NHOSAC) is a Japanese hospital-based cohort study investigating the safety of the initial use of glucocorticoids (GCs) in patients with newly diagnosed autoimmune diseases. Using the J-NHOSAC registry, the purpose of this observational study is to analyse the rates, characteristics and associated risk factors of intracellular infections in patients with newly diagnosed autoimmune diseases who were initially treated with GCs.

Methodology/Principal Findings

A total 604 patients with newly diagnosed autoimmune diseases treated with GCs were enrolled in this registry between April 2007 and March 2009. Cox proportional-hazards regression was used to determine independent risk factors for serious intracellular infections with covariates including sex, age, co-morbidity, laboratory data, use of immunosuppressants and dose of GCs. Survival was analysed according to the Kaplan-Meier method and was assessed by the log-rank test. There were 127 serious infections, including 43 intracellular infections, during 1105.8 patient-years of follow-up. The 43 serious intracellular infections resulted in 8 deaths. After adjustment for covariates, diabetes (Odds ratio [OR]: 2.5, 95% confidence interval [95% CI] 1.1–5.9), lymphocytopenia (≦1000/μl, OR: 2.5, 95% CI 1.2–5.2) and use of high-dose (≧30 mg/day) GCs (OR: 2.4, 95% CI 1.1–5.3) increased the risk of intracellular infections. Survival curves showed lower intracellular infection-free survival rate in patients with diabetes, lymphocytopaenia and high-dose GCs treatments.

Conclusions/Significance

Patients with newly diagnosed autoimmune diseases were at high risk of developing intracellular infection during initial treatment with GCs. Our findings provide background data on the risk of intracellular infections of patients with autoimmune diseases. Clinicians showed remain vigilant for intracellular infections in patients with autoimmune diseases who are treated with GCs.     

Somatic Donor Cell Type Correlates with Embryonic,but Not Extra-Embryonic,Gene Expression in Postimplantation Cloned Embryos

The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (>2-fold vs. controls) than did the extraembryonic tissues (P<1.0×10^–26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1–5% per embryos transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.     

Phytotoxic Metabolites of Phyllosticta sp.

The additional new metabolites, named phyllostine (II) and 3-chloro-2,5-dihydroxybenzyl alcohol (III) from the cultural filtrates of Phyllosticta sp. have been isolated. The chemical structure of II have been established by spectral studies and chemical conversion from the known I, and the chlorine-containing metabolite (III) by spectral and synthetic studies. The metabolite (II) exhibits the similar phytotoxic effects as I, but the metabolite III does less phytotoxicity than I and II on the leaf test.     

Studies on the Polyuronide of Oh-gyo-tye (The Achine of Ficus awkeotsang MAKINO)

A polyuronide, main component of the water extract of achine of Ficus awkeotsang MAKINO (on-gyo-tye), was purified by ion-exchange chromatography on DEAE-cellulose. The polyuronide (Fraction IB) is homogeneous electrophoretically and consists mainly of galacturonic acid. Optical rotation of Fraction IB is and content of methoxyl group is trace. In periodate oxidation of Fraction IB, molar ratio of galacturonic acid residue and periodate consumption was 1, and formic acid formation was very small. Periodate oxidation product of Fraction IB was oxidized further with bromine and the resulted substance was hydrolyzed. In the hydrolyzate, presence of large amount of tartaric and glyoxylic acids and small amount of tartronic acid were detected by paper chromatography. Reduced viscosity of aquous solution of Fraction IB increased with decreasing of the concentration of Fraction IB solution. From these results, it was deduced that Fraction IB has a linear structure of 1→4 linkage of d-galacturonic acid, probably α-linkage.     

Isolation and Structure of Phytotoxic Compounds Produced by Phyllosticta sp.

In the course of phytopathological studies, it has been observed that phyllosticta sp. produces a toxic compound causing wilting and simultaneous dark coloration of the clover leaf. A toxin, herein refered to as phyllosinol (mp 76~77°C) was isolated in a crystalline state from the pure culture and it was proved to be the principal toxic metabolite of the fungus. Although the melting point of phyllosinol differed significantly from that reported for epoxydon (40~45°C), the structural evidence deduced from spectrometric and chemical methods indicates that phyllosinol is substantially identical with epoxydon even in stereo-isomeric considerations. In the red clover leaf test 10 ppm phyllosinol showed the wilting effect.     

miR-146a Polymorphism (rs2910164) Predicts Colorectal Cancer Patients’ Susceptibility to Liver Metastasis

miR-146a plays important roles in cancer as it directly targets NUMB, an inhibitor of Notch signaling. miR-146a is reportedly regulated by a G>C polymorphism (SNP; rs2910164). This polymorphism affects various cancers, including colorectal cancer (CRC). However, the clinical significance of miR-146a polymorphism in CRC remains unclear. A total of 59 patients with CRC were divided into 2 groups: a CC/CG genotype (n = 32) and a GG genotype (n = 27), based on the miR-146a polymorphism. cDNA microarray analysis was performed using 59 clinical samples. Significantly enriched gene sets in each genotype were extracted using GSEA. We also investigated the association between miR-146a polymorphism and miR-146a, NUMB expression or migratory response in CRC cell lines. The CC/CG genotype was associated with significantly more synchronous liver metastasis (p = 0.007). A heat map of the two genotypes showed that the expression profiles were clearly stratified. GSEA indicated that Notch signaling and JAK/STAT3 signaling were significantly associated with the CC/CG genotype (p = 0.004 and p = 0.023, respectively). CRC cell lines with the pre-miR-146a/C revealed significantly higher miR-146a expression (p = 0.034) and higher NUMB expression and chemotactic activity. In CRC, miR-146a polymorphism is involved in liver metastasis. Identification of this polymorphism could be useful to identify patients with a high risk of liver metastasis in CRC.