Cloning and characterization of the lactate dehydrogenase genes from<Emphasis Type="Italic">Lactobacillus</Emphasis> sp. RKY2

Lactic acid is an environmentally benign organic acid that could be used as a raw material for biodegradable plastics if it can be inexpensively produced by fermentation. Two genes (IdhL andIdhD) encoding the L-(+) and D-(−) lactate dehydrogenases (L-LDH and D-LDH) were cloned fromLactobacillus sp., RKY2, which is a lactic acid hyper-producing bacterium isolated from Kimchi. Open reading frames ofIdhL for andIdhD for the L and D-LDH genes were 962 and 998 bp, respectively. Both the L(+)- and D(−)-LDH proteins showed the highest degree of homology with the L- and D-lactate dehydrogenase genes ofLactobacillus plantarum. The conserved residues in the catalytic activity and substrate binding of both LDHs were identified in both enzymes.     

Group IB secretory phospholipase A2 promotes matrix metalloproteinase-2-mediated cell migration via the phosphatidylinositol 3-kinase and Akt pathway

Secretory phospholipase A(2) (sPLA(2)), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA(2) increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA(2) in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA(2) decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1-MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA(2)-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H(+)-ATPase, sustained MMP-2 activation by sPLA(2). The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA(2). Expression of p85alpha and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA(2)-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA(2) increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA(2)-promoted fibroblasts migration.     

Investigation of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli in Korea

AIMS: Isolates obtained from various regions in Korea in 2002 were identified and their susceptibility to extended-spectrum cephalosporins, monobactams and/or cephamycins was studied along with any production of extended-spectrum beta-lactamases (ESBLs). METHODS AND RESULTS: Bacteria identified by the conventional techniques and Vitek GNI card were Klebsiella pneumoniae and Escherichia coli. Using disk diffusion and double-disk synergy tests, we found that 39.2% of strains produced ESBLs. About 52% of isolates transferred resistance to ceftazidime by conjugation. Banding patterns of PCR amplification with the designed primers showed that 837- and 259-bp fragments specific to bla(TEM) genes were amplified in 63.3% of strains. 929- and 231-bp fragments (bla(SHV)), 847- and 520-bp fragments (bla(CMY)), 597- and 858-bp fragments (bla(CTX-M)) were amplified in 61.5, 17.3 and 7.7% of strains respectively. About 51.9% of strains contained more than two types of beta-lactamase genes. Especially, one strain contained bla(TEM), bla(CMY) and bla(CTX-M) genes. SIGNIFICANCE: Resistance mechanisms to beta-lactams, comprising mostly ESBL production, lead to the resistance against even recently developed beta-lactams in enterobacteria, which is now a serious threat to antibiotic therapy. The high prevalence of bla(CMY) genes and multidrug-resistant genes may also make therapeutic failure and lack of eradiation of these strains by extended-spectrum cephalosporins or cephamycins.     

Novel role for Netrins in regulating epithelial behavior during lung branching morphogenesis

The development of many organs, including the lung, depends upon a process known as branching morphogenesis, in which a simple epithelial bud gives rise to a complex tree-like system of tubes specialized for the transport of gas or fluids. Previous studies on lung development have highlighted a role for fibroblast growth factors (FGFs), made by the mesodermal cells, in promoting the proliferation, budding, and chemotaxis of the epithelial endoderm. Here, by using a three-dimensional culture system, we provide evidence for a novel role for Netrins, best known as axonal guidance molecules, in modulating the morphogenetic response of lung endoderm to exogenous FGFs. This effect involves inhibition of localized changes in cell shape and phosphorylation of the intracellular mitogen-activated protein kinase(s) (ERK1/2, for extracellular signal-regulated kinase-1 and -2), elicited by exogenous FGFs. The temporal and spatial expression of netrin 1, netrin 4, and Unc5b genes and the localization of Netrin-4 protein in vivo suggest a model in which Netrins in the basal lamina locally modulate and fine-tune the outgrowth and shape of emergent epithelial buds.     

Antimicrobial and cytotoxic activities of neolignans from Magnolia officinalis

In the light of the steady increase of infections related to vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), the medicinal plant Magnolia officinalis was subjected to bioassay-directed fractionation, which led to the isolation of the known neolignans piperitylmagnolol (1), magnolol (2), and honokiol (3) from the MeOH extract. In broth-microdilution assays, 1-3 exhibited antibacterial activities against VRE and MRSA at minimum-inhibitory concentrations (MIC) in the range of 6.25-25 microg/ml, compound 1 being the most-potent antibiotic. The ratio of MBC/MIC (MBC = minimum bactericidal concentration) was < or = 2 for all compounds. The kinetics of the antibacterial action of 1 and 3 were studied by means of time-kill assays; both compounds were bactericidal against VRE and MRSA, their actions being time dependent, or both time and concentration dependent. Magnolol (2) was acetylated to magnolol monoacetate (4) and magnolol diacetate (5) (partial or full masking of the phenolic OH functions). The cytotoxic properties of 1-5 against human OVCAR-3 (ovarian adenocarcinoma), HepG2 (hepatocellular carcinoma), and HeLa (cervical epitheloid carcinoma) cell lines were evaluated. The CD50 values for compounds 1-3 were in the range of 3.3-13.3 microg/ml, derivatives 4 and 5 being much less potent. This study indicates that piperitylmagnolol (= 3-[(1S,6S)-6-isopropyl-3-methylcyclohex-2-enyl]-5,5'-di(prop-2-enyl)[1,1'-biphenyl]-2,2'-diol; 1) possesses both significant anti-VRE activity and moderate cytotoxicity against the above cancer cell lines.     

A comparative analysis of the complete mitochondrial genome of the Eurasian otter <Emphasis Type="Italic">Lutra lutra</Emphasis> (Carnivora; Mustelidae)

Otter populations are declining throughout the world and most otter species are considered endangered. Molecular methods are suitable tools for population genetic research on endangered species. In the present study, we analyzed the complete mitochondrial genome (mitogenome) sequence of the Eurasian otter Lutra lutra. The mitochondrial DNA sequence of the Eurasian otter is 16,505 bp in length and consists of 13 protein-coding genes, 22 tRNAs, 2 rRNAs, and a control region (CR). The CR sequence of otters from Europe and Asia showed nearly identical numbers and nucleotide sequences of minisatellites. Phylogenetic analysis of Mustelidae mitogenomes, including individual genes, revealed that Lutrinae and Mustelinae form a clade, and that L. lutra and Enhydra lutris are sister taxa within the Lutrinae. Phylogenetic analyses revealed that of the 13 mitochondrial protein-coding genes, ND5 is the most reliable marker for analysis of phylogenetic relationships within the Mustelidae.     

Ala99ser mutation in RIα Regulatory Subunit of protein kinase A causes reduced kinase activation by cAMP and arrest of hormone-dependent breast cancer cell growth

Expression of the RI regulatory subunit of protein kinase A type I is increased in human cancer cell lines, in primary tumors, in cells after transformation, and in cells upon stimulation of growth. Ala99 (the pseudophosphorylation site) of human RI was replaced with Ser (RI-p) for the structure-function analysis of RI. MCF-7 hormone- dependent breast cancer cells were transfected with an expression vector for the wild-type RI or mutant RI-p. Overexpression of RI-P resulted in suppression of protein kinase A type II, the isozyme of type I kinase, production of kinase exhibiting reduced cAMP activation, and inhibition of cell growth showing an increase in G0/G1 phase of the cell cycle and apoptosis. The wild-type RI overexpression had no effect on protein kinase A isozyme distribution or cell growth. Overexpression of protein kinase A type II regulatory subunit, RII, suppressed RI and protein kinase A type I and inhibited cell growth. These results show that the growth of hormone-dependent breast cancer cells is dependent on the functional protein kinase A type I.     

A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.