Fumarate-Mediated Persistence of Escherichia coli against Antibiotics.

Bacterial persisters are a small fraction of quiescent cells that survive in the presence of lethal concentrations of antibiotics. They can regrow to give rise to a new population that has the same vulnerability to the antibiotics as did the parental population. Although formation of bacterial persisters in the presence of various antibiotics has been documented, the molecular mechanisms by which these persisters tolerate the antibiotics are still controversial. We found that amplification of the fumarate reductase operon (FRD) inEscherichia coliled to a higher frequency of persister formation. The persister frequency ofE. coliwas increased when the cells contained elevated levels of intracellular fumarate. Genetic perturbations of the electron transport chain (ETC), a metabolite supplementation assay, and even the toxin-antitoxin-relatedhipA7mutation indicated that surplus fumarate markedly elevated theE. colipersister frequency. AnE. colistrain lacking succinate dehydrogenase (SDH), thereby showing a lower intracellular fumarate concentration, was killed ∼1,000-fold more effectively than the wild-type strain in the stationary phase. It appears thatSDHandFRDrepresent a paired system that gives rise to and maintainsE. colipersisters by producing and utilizing fumarate, respectively.

ATHB12, an ABA-inducible homeodomain-leucine zipper (HD-Zip) protein of Arabidopsis, negatively regulates the growth of the inflorescence stem by decreasing the expression of a gibberellin 20-oxidase gene.

Arabidopsis thaliana homeobox 12 (ATHB12) is rapidly induced by ABA and water stress. A T-DNA insertion mutant of ATHB12 with a reduced level of ATHB12 expression in stems had longer inflorescence stems and reduced sensitivity to ABA during germination. A high level of transcripts of gibberellin 20-oxidase 1 (GA20ox1), a key enzyme in the synthesis of gibberellins, was detected in athb12 stems, while transgenic lines overexpressing ATHB12 (A12OX) had a reduced level of GA20ox1 in stems. Consistent with these data, ABA treatment of wild-type plants resulted in decreased GA20ox1 expression whereas ABA treatment of the athb12 mutant gave rise to slightly decreased GA20ox1 expression. Retarded stem growth in 3-week-old A12OX plants was rescued by exogenous GA(9), but not by GA(12), and less GA(9) was detected in A12OX stems than in wild-type stems. These data imply that ATHB12 decreases GA20ox1 expression in stems. On the other hand, the stems of A12OX plants grew rapidly after the first 3 weeks, so that they were almost as high as wild-type plants at about 5 weeks after germination. We also found changes in the stems of transgenic plants overexpressing ATHB12, such as alterations of expression GA20ox and GA3ox genes, and of GA(4) levels, which appear to result from feedback regulation. Repression of GA20ox1 by ATHB12 was confirmed by transfection of leaf protoplasts. ABA-treated protoplasts also showed increased ATHB12 expression and reduced GA20ox1 expression. These findings all suggest that ATHB12 negatively regulates the expression of a GA 20-oxidase gene in inflorescence stems.

An RNA-hydrolyzing recombinant antibody exhibits an antiviral activity against classical swine fever virus.

Some proteins with ribonuclease (RNase) activity have been shown to suppress viral replication. A well-characterized recombinant antibody, 3D8 single-chain variable fragment (3D8 scFv), has RNA-hydrolyzing and cell-penetrating activities. Here, we investigated antiviral activity of 3D8 scFv against classical swine fever virus (CSFV). In a cell line expressing 3D8 scFv (C26), intracellular RNA-hydrolysis activity was higher compared to control PK-15 cells and viral replication was strongly suppressed at the viral RNA level, with the evidence of independency of IFN-alpha/beta induction. Exogenous treatment of 3D8 scFv, prior to or post-CSFV infection, was also shown to suppress CSFV replication at the viral RNA level. These observations suggest that antiviral activity of 3D8 scFv may be due to the intrinsic RNase activity of 3D8 scFv, which is capable of targeting viral RNA genomes or transcripts.

Overexpression of the downward leaf curling (DLC) gene from melon changes leaf morphology by controlling cell size and shape in Arabidopsis leaves.

A plant-specific gene was cloned from melon fruit. This gene was named downward leaf curling (CmDLC) based on the phenotype of transgenic Arabidopsis plants overexpressing the gene. This expression level of this gene was especially upregulated during melon fruit enlargement. Overexpression of CmDLC in Arabidopsis resulted in dwarfism and narrow, epinastically curled leaves. These phenotypes were found to be caused by a reduction in cell number and cell size on the adaxial and abaxial sides of the epidermis, with a greater reduction on the abaxial side of the leaves. These phenotypic characteristics, combined with the more wavy morphology of epidermal cells in overexpression lines, indicate that CmDLC overexpression affects cell elongation and cell morphology. To investigate intracellular protein localization, a CmDLC-GFP fusion protein was made and expressed in onion epidermal cells. This protein was observed to be preferentially localized close to the cell membrane. Thus, we report here a new plant-specific gene that is localized to the cell membrane and that controls leaf cell number, size and morphology.

Comprehensive analysis of microRNA-mRNA co-expression in circadian rhythm.

To investigate the potential role of microRNA (miRNA) in the regulation of circadian rhythm, we performed microarray-based expression profiling study of both miRNA and mRNA in mouse liver for 48 h at 4-hour intervals. Circadian miRNA-mRNA target pair is defined as the pair both elements of which show circadian expression patterns and the sequence-based target relationship of which can be predicted. Circadian initiators, Clock and Bmal1, showed inversely correlated circadian expression patterns against their corresponding miRNAs, miR-181d and miR-191, targeting them. In contrast, circadian suppressors, Per, Cry, CKIe and Rev-erba, exhibited positively correlated circadian expression patterns to their corresponding miRNAs. Genomic location analysis revealed that intronic region showed higher abundance of cyclic than non-cyclic miRNAs targeting circadian genes while other (i.e., 3-UTR, exon and intergenic) regions showed no difference. It is suggested that miRNAs are involved in the regulation of peripheral circadian rhythm in mouse liver by modulating Clock:Bmal1 complex. Identifying specific miRNAs and their targets that are critically involved in circadian rhythm will provide a better understanding of the regulation of circadian- clock system.

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting.

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.

Recombinant DNA and Protein Vaccines for Foot-and-mouth Disease Induce Humoral and Cellular Immune Responses in Mice.

BACKGROUND: Foot-and-mouth disease virus (FMDV) is a small single-stranded RNA virus which belongs to the family Picornaviridae, genus Apthovirus. It is a principal cause of FMD which is highly contagious in livestock. In a wild type virus infection, infected animals usually elicit antibodies against structural and non-structural protein of FMDV. A structural protein, VP1, is involved in neutralization of virus particle, and has both B and T cell epitopes. A RNA-dependent RNA polymerase, 3D, is highly conserved among other serotypes and strongly immunogenic, therefore, we selected VP1 and 3D as vaccine targets. METHODS: VP1 and 3D genes were codon-optimized to enhance protein expression level and cloned into mammalian expression vector. To produce recombinant protein, VP1 and 3D genes were also cloned into pET vector. The VP1 and 3D DNA or proteins were co-immunized into 5 weeks old BALB/C mice. RESULTS: Antigen-specific serum antibody (Ab) responses were detected by Ab ELISA. Cellular immune response against VP1 and 3D was confirmed by ELISpot assay. CONCLUSION: The results showed that all DNA- and protein-immunized groups induced cellular immune responses, suggesting that both DNA and recombinant protein vaccine administration efficiently induced Ag-specific humoral and cellular immune responses.

Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity.

Anti-DNA antibodies (Abs) are of biomedical interest because they are associated with autoimmune diseases in human and mice. Previously we isolated an anti-DNA monoclonal Ab 3D8 from an autoimmune-prone MRL-lpr/lpr mouse. Here we have characterized DNA binding kinetics and hydrolyzing activities of the recombinant single chain variable fragment (scFv) and the single variable domains of heavy chain (VH) and light chain (VL) using various single-stranded (ss) and double-stranded (ds) DNA substrates. All the Abs bound to both ds- and ssDNAs without significant preferential sequence specificity showing scFv higher affinities (KD = approximately 17-74 nm) than VH (KD = approximately 2.4-8.4 microm) and VL (KD = approximately 3.2-72 microm), and efficiently hydrolyzed both ds- and ssDNAs without sequence specificity in a Mg2+-dependent manner, except for the poor activity of 3D8 scFv for ss-(dT)40. Elucidated crystal structure-based His to Ala mutations on the complementarity determining regions of VH (His-H35 --> Ala) and/or VL (His-L94 --> Ala) of 3D8 scFv significantly inhibited the catalytic activities, indicating that the His residues are involved in the catalytic mechanism of 3D8 scFv. However, the DNA hydrolyzing activities of single domain VH and VL were not affected by the mutations, indicative of their different catalytic mechanisms from that of 3D8 scFv. Our results demonstrate single domain Abs with DNase activities for the first time, which might provide new insights into substrate recognition and catalytic mechanisms of anti-DNA Abs.