Complete Genome Sequence Analysis of Leuconostoc kimchii IMSNU 11154

Leuconostoc kimchii IMSNU 11154, isolated from kimchi, a traditional Korean fermented food, is known to be an important antimicrobial lactic acid bacterium with probiotic potential. Here we announce the complete genome sequence of L. kimchii IMSNU 11154 consisting of a 2,101,787-bp chromosome and five plasmids. The strain has genes for dextran formation from sucrose and for mannitol formation from fructose. Antimicrobial and antioxidative functions of L. kimchii IMSNU 11154 could be attributed to a leucosin B-like peptide and multiple enzymes to reduce hydrogen peroxide and oxidized thiols, respectively.Kimchi is a traditional Korean pickled vegetable dish with varied seasonings, and it is known to be an important source of vitamins, minerals, and dietary fiber as well as a good dietary source of lactic acid bacteria (LAB) for humans (2, 3). An exopolysaccharide (EPS)-producing LAB, designated IMSNU 11154, was isolated from kimchi made of cabbage and subsequently classified as a novel species, Leuconostoc kimchii (6). The strain and its culture broth showed antimicrobial activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus cepacia, Candida albicans, and Aspergillus niger. Here we report the genome sequence of Leuconostoc kimchii IMSNU 11154 using a whole-genome shotgun sequencing strategy (4). The complete sequences of one chromosome and five plasmids were obtained. The circular chromosome of strain IMSNU 11154 was 2,101,787 bp in length (37.9% G+C), and the five plasmids were LkipL48 (3,196 bp; 37.1% G+C), LkipL4701 (21,055 bp; 34.3% G+C), LkipL4704 (23,285 bp; 35.6% G+C), LkipL4719 (21,924 bp; 39.1% G+C), and LkipL4726 (29,616 bp; 35.5% G+C). The number of predicted open reading frames (ORFs) is 2,205, covering 89.5% (1,880,952 bp) of the genome. Noncoding RNA regions were identified as 68 tRNAs and 4 copies of rRNA operons. A small gene encoding a leucosin B-like peptide was identified.Strain IMSNU 11154 was missing the fructose 1,6-bisphosphatase enzyme of the Embden-Meyerhof-Parnas pathway and transaldolase of the 6-phosphogluconate/phosphoketolase pathway. Tricarboxylic cycle enzymes were also absent, except for cytochrome oxidase bd-I subunits. The strain metabolized sucrose by invertase, sucrose phosphorylase, and dextransucrase. Formation of EPS and fructose from sucrose by dextransucrase could enhance the probiotic function as well as improve the taste and flavor of kimchi. Strain IMSNU 11154 had a mannitol dehydrogenase gene (mdh) identical to mdh of Leuconostoc mesenteroides (1). By producing mannitol, it maintains its turgor and stabilizes membrane lipids and proteins at low water activity as well as scavenges free reactive oxygen radicals as previously observed for mannitol fermenters (10). Like other Leuconostoc spp., strain IMSNU 11154 does not bear any catalase or superoxide dismutase (SOD) enzymes but has six thioredoxins and four thioredoxin reductases that were shown to be important in coping with acid and oxidative stress (9, 11). Harboring thioredoxin systems on plasmids is a common feature for both L. kimchii IMSNU 11154 and Leuconostoc citreum KM20 (7). Glutathione protected some lactic acid bacteria against oxidative stress (8), but gamma-glutamylcysteine (γ-GC) is the major low-molecular-weight thiol in Leuconostoc spp., including IMSNU 11154 (5). Genes for γ-GC synthesis and reduction are present in strain IMSNU 11154 (5), and there are genes for putative peroxiredoxins that can reduce hydrogen peroxide via small thiols or thioredoxins. Thus, multiple antioxidative systems reduce thiols in strain IMSNU 11154.In conclusion, the genome of Leuconostoc kimchii IMSNU 11154 revealed that its carbohydrate metabolism has adapted to the formation of dextran, fructose, and mannitol from sucrose. The antimicrobial activity of strain IMSNU 11154 could be due to a leucosin B-like peptide, and it contains multiple antioxidative systems to manage acid and oxidative stresses independent of SOD and catalase.     

Regulation of TonEBP transcriptional activator in MDCK cells following changes in ambient tonicity

In response to ambient hypertonicity, TonEBP (tonicity-responsive enhancer binding protein) stimulates certain genes including those encoding cytokines, transporters for organic solutes, and a molecular chaperone. TonEBP is regulated in a bidirectional manner, upregulated by an increase in ambient tonicity while downregulated by a decrease. To investigate the role of intracellular ionic strength in the activity of TonEBP, we subjected Madin-Darby canine kidney cells to a variety of conditions. Electron microprobe analysis was performed to measure intracellular electrolytes. Under conditions in which changes in cell volume were similar, TonEBP activity correlated with the intracellular ionic strength regardless of the external tonicity. On the other hand, inhibition of the Na+/K+-ATPase and high external K+ concentration led to a decreased activity of TonEBP despite a marked increase in the intracellular ionic strength. Because isotonic swelling is known to occur under these conditions, these data suggest that dilution of the cytoplasmic constituents inhibits the activity of TonEBP. We conclude that intracellular ionic strength and water content are major factors that determine the activity of TonEBP.     

Quinolone compounds with carboxylic equivalents and semiempirical calculations on their tautomers

Quinolone antibiotic compounds having no carboxylic group but its equivalents at the 3-position have been synthesized. Energies of tautomers with the carboxylic mimics were calculated in gas phase and aqueous solution with AM1-SCRF method. Antibacterial activity of the compounds was rationalized with tautomeric protons and atomic charges of calculated tautomers.     

Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

Significance of N-methyl-D-aspartate (NMDA) receptor-mediated signaling in human keratinocytes

Increasing data suggest that glutamate might act as a cell-signaling molecule in non-neuronal tissues such as the skin. Here we demonstrate the presence of functional N-methyl-D-aspartate (NMDA)-type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell-to-cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re-epithelialization, and the process of re-epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact-mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.     

Differential expression of T-type calcium channels in P/Q-type calcium channel mutant mice with ataxia and absence epilepsy

Mutations in P/Q-type calcium channels generate common phenotypes in mice and humans, which are characterized by ataxia, paroxysmal dyskinesia, and absence seizures. Subsequent functional changes of T-type calcium channels in thalamus are observed in P/Q-type calcium channel mutant mice and these changes play important roles in generation of absence seizures. However, the changes in T-type calcium channel function and/or expression in the cerebellum, which may be related to movement disorders, are still unknown. The leaner mouse exhibits severe ataxia, paroxysmal dyskinesia, and absence epilepsy due to a P/Q-type calcium channel mutation. We investigated changes in T-type calcium channel expression in the leaner mouse thalamus and cerebellum using quantitative real-time polymerase chain reaction (qRT-PCR) and quantitative in situ hybridization histochemistry (ISHH). qRT-PCR analysis showed no change in T-type calcium channel alpha 1G subunit (Cav3.1) expression in the leaner thalamus, but a significant decrease in alpha 1G expression in the whole leaner mouse cerebellum. Interestingly, quantitative ISHH revealed differential changes in alpha 1G expression in the leaner cerebellum, where the granule cell layer showed decreased alpha 1G expression while Purkinje cells showed increased alpha 1G expression. To confirm these observations, the granule cell layer and the Purkinje cell layer were laser capture microdissected separately, then analyzed with qRT-PCR. Similar to the observation obtained by ISHH, the leaner granule cell layer showed decreased alpha 1G expression and the leaner Purkinje cell layer showed increased alpha 1G expression. These results suggest that differential expression of T-type calcium channels in the leaner cerebellum may be involved in the observed movement disorders.     

Ecophysiology of selected tree species in different plant communities at the periphery of the Atlantic Forest of SE Brazil I. Performance of three different species of Clusia in an array of plant communities

Three species of Clusia, namely two CAM-species (C. hilariana Schlecht. and C. fluminensis Planch. et Triana) and a C₃-species (C. parviflora Saldanha et Engl.) were studied in different plant communities at the periphery of the Atlantic Forest in the state of Rio de Janeiro, Brazil. The sites chosen were an inselberg (C. parviflora), a range of wet restinga – dry restinga – semideciduous dry forest (C. fluminensis), and a gradient from the sea shore inland with a first sand dune beach ridge – a dry forest – a second beach ridge (C. hilariana). Analyses comprised C and N contents, soluble carbohydrates, soluble nonprotein N compounds and carbon isotope ratios (¹³C) in leaves, roots, phloem and wood. Photosynthetic performance was assessed by chlorophyll fluorescence with measurements of instant photosynthetic yield as well as light dependence and potential quantum-use efficiency of photosystem II.The data allow, first, to discuss differences between the ecophysiological performance of C₃- and CAM-species of Clusia. The C₃-species, C. parvifolia, had an overall weaker performance than the two CAM-species, where, however, the effects of mode of photosynthesis may have been overlaid by site conditions. Second, it was studied whether ecophysiological performance relates to patterns of local abundance, which was confirmed by showing that the dominant Clusia species of the restingas, C. hilariana, showed the strongest performance overall.Finally, it was studied whether the ecophysiological performance varied in response to site-dependent gradients of environmental water relations, which was confirmed for functions such as photosynthetic capacity, photoinhibition and solute accumulation of C. hilariana and C. fluminensis in relation to moisture of sites.