Generation and Characterization of Thymidine/d-Alanine Auxotrophic Recombinant Lactococcus lactis subsp. lactis IL1403 Expressing BmpB

Genetic engineering of Lactococcus lactis to produce a heterologous protein may cause potential risks to the environment despite the industrial usefulness of engineered strains. To reduce the risks, we generated three auxotrophic recombinant L. lactis subsp. lactis IL1403 strains expressing a heterologous protein, BmpB, using thyA- and alr-targeting integration vectors: ITD (thyA ⁻ alr ⁺ bmpB ⁺), IAD (thyA ⁺ alr ⁻ bmpB ⁺), and ITDAD (thyA ⁻ alr ⁻ bmpB ⁺). After construction of integration vectors, each vector was introduced into IL1403 genome. Integration of BmpB expression cassette, deletion of thyA, and inactivation of alr were verified by using PCR reaction. All heterologous DNA fragments except bmpB were eliminated from those recombinants during double crossover events. By using five selective agar plates, we also showed thymidine auxotrophy of ITD and ITDAD and d-alanine auxotrophy of IAD and ITDAD. In M17G and skim milk (SYG) media, the growth of the three recombinants was limited. In MRS media, the growth of IAD and ITDAD was limited, but ITD showed a normal growth pattern as compared with the wild-type strain (WT). All the recombinants showed maximal BmpB expression at an early stationary phase when they were cultivated in M17G supplemented with thymidine and d-alanine. These results suggest that auxotrophic recombinant L. lactis expressing a heterologous protein could be generated to reduce the ecological risks of a recombinant L. lactis.     

Triad 1 induces apoptosis by p53 activation

Triad 1 (2 RING [really interesting new gene] fingers and DRIL [double RING finger linked] 1) is an E3 ligase that induces apoptosis and clonogenic inhibition in myeloid cells through Gfi-1 stabilization. Here we demonstrate that Triad 1 induces apoptosis in several cancer cell lines including MCF7, A549, U2OS, and HCT 116 p53^+/+ cells via its RING ligase activity. Interestingly, in these cancer cells, Triad 1-induced apoptosis is not mediated by Gfi-1 stabilization but is instead p53-dependent. Moreover, Triad 1 promotes transactivation of p53. These results suggest that Triad 1 can induce apoptosis through its ligase activity via p53 activation.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

NM23-H2 involves in negative regulation of Diva and Bcl2L10 in apoptosis signaling

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. Diva (Boo), an ortholog of Bcl2L10 or Bcl-B, is a member of the Bcl-2 family that has contradictory functions in apoptosis. To understand the signaling mechanisms of Diva, we searched for proteins that interact with Diva using the yeast two-hybrid system. We identified a nucleoside diphosphate kinase isoform, NM23-H2. Here, we show that Diva bound to NM23-H2 in cells in which the transmembrane domain of Diva was required, and both proteins were colocalized in cytoplasm. Of interest, Diva protein level was significantly down-regulated by NM23-H2 as knock down of NM23-H2 restored Diva expression. Overexpression of NM23-H2 induced apoptosis, and the depletion of NM23-H2 led to the increase of Diva's apoptotic activity. Thus, these results indicate the existence of a previously undiscovered mechanism by which NM23-H2 involves in the regulation of Diva-mediated apoptosis.     

Autolysis loop restricts the specificity of activated protein C: analysis by FRET and functional assays

We previously demonstrated that the substitution of the autolysis loop (residues 143-154 in chymotrypsin numbering) of APC with the corresponding loop of trypsin (APC-Tryp 143-154) has no influence on the proteolytic activity of the protease toward fVa, however, this substitution increases the reactivity of APC with plasma inhibitors so that the mutant exhibits no anticoagulant activity in plasma. To further investigate the role of the autolysis loop in APC and determine whether this loop is a target for modulation by protein S, we evaluated the activity of APC-Tryp 143-154 toward fVa and several plasma inhibitors both in the absence and presence of protein S. Furthermore, we evaluated the active-site topography of APC-Tryp 143-154 by determining the average distance of the closest approach (L) between a fluorescein dye tethered to a tripeptide inhibitor, attached to the active-site of APC-Tryp 143-154, and octadecylrhodamine dyes incorporated into PCPS vesicles both in the absence and presence of protein S. The activity of APC-Tryp 143-154 toward fVa was identical to that of wild-type APC both in the presence and absence of protein S. However, the reactivity of APC-Tryp 143-154 with plasma inhibitors was preferentially improved independent of protein S. The FRET analysis revealed a dramatic change in the active-site topography of APC both in the absence and presence of protein S. Anisotropy measurements revealed that the fluorescein dye has a remarkable degree of rotational freedom in the active-site of APC-Tryp 143-154. These results suggest that the autolysis loop of APC may not be a target for modulation by protein S. This loop, however, plays a critical role in restricting both the specificity and spatial environment of the active-site groove of APC.     

Construction and characterization of a recombinant whole-cell biocatalyst of Escherichia coli expressing styrene monooxygenase under the control of arabinose promoter

A recombinant Escherichia coli (pBAB1) containing styrene monooxygenase (SMO) was developed for the conversion of styrene to enantiopure (S)-styrene oxide that is an important chiral building block in organic synthesis. The styAB genes encoding SMO was cloned into a multicopy plasmid under the tightly regulated promoter of bacterial l-arabinose operon which is inducible by l-arabinose. The recombinant showed that expression level of StyA protein and whole-cell SMO activities were varied depending on the concentration of the inducer l-arabinose. The maximum SMO activity was 108 U/g cdw when the cells were induced with 0.2% l-arabinose. SDS-PAGE and Western blot analyses indicated that whole-cell SMO activity was strongly correlated with the expression level of StyA protein. Organic-aqueous two-phase experiment could yield 50 mM enantiopure (S)-styrene oxide in organic phase in 18 h, but the recombinant SMO activity was unstable during the reaction. The expression of styAB under the control of l-arabinose promoter was significantly repressed in the presence of glucose.     

Anatomy and ultrastructure of epidermal cells in the haustorium of a parasitic flowering plant,Cuscuta japonica, during attachment to the host

Changes of epidermal cells in the haustorium of the parasiticCuscuta japonica during its attachment to the host plantimpatiens balsamina were studied with light and electron microscopy. In the transverse sections of dodder stems not in contact with the host, epidermal cells had rounded outlines. However, when haustorial initials developed in the cortex of the parasite stem at the contact site, the epidermal cells had more dense cytoplasm and conspicuous nuclei than before, and their outline was flat in the longitudinal section. As meristem cells developed from those initials, the epidermal cells became more elongated. When the haustorium was fully matured, the apical tips of the elongated epidermal cells at the contact site branched like toes, producing numerous projections via cell wall invaginations. This event caused spaces to form between the projections; coincidently, the surface area of the apical ends of the epidermal cells increased. The dense cytoplasm at those projections contained prominent nuclei and abundant other organelles, suggesting a active metabolism. Osmiophilic particles, releasing into the cell walls from the cytoplasm, were though to be associated with the loosening and elongating of the epidermal cell walls. Dense and homogeneous materials were secreted within the spaces between the projections. These materials could play an important role in cementing the haustorium onto the surface of the host organ.     

<Emphasis Type="Italic">Luteimonas aestuarii</Emphasis> sp. nov., isolated from tidal flat sediment

A novel bacterium B9^T was isolated from tidal flat sediment. Its morphology, physiology, biochemical features, and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow and the cells are Gram-negative, rod-shaped, and do not require NaCl for growth. The 16S rRNA gene sequence similarity indicated that strain B9^T is associated with the genus Lysobacter (≤ 97.2%), Xanthomonas (≤ 96.8%), Pseudomonas (≤ 96.7%), and Luteimonas (≤ 96.0%). However, within the phylogenetic tree, this novel strain shares a branching point with the species Luteimonas composti CC-YY255^T (96.0%). The DNA-DNA hybridization experiments showed a DNA-DNA homology of 23.0% between strain B9^T and Luteimonas mephitis B1953/27.1^T. The G+C content of genomic DNA of the type strain is 64.7 mol% (SD, 1.1). The predominant fatty acids are iso-C_11:0, iso-C_15:0, iso-C_16:0, iso-C_17:0, iso-C_17:0 ω9c, and iso-C_11:0 3-OH. Combined analysis of the 16S rRNA gene sequences, fatty acid profile, and results from physiological and biochemical tests indicated that there is genotypic and phenotypic differentiation of the isolate from other Luteimonas species. For these reasons, strain B9^T was proposed as a novel species, named Luteimonas aestuarii. The type strain of the new species is B9^T (= KCTC 22048^T, DSM 19680^T).     

Linker region of Akt1/protein kinase Balpha mediates platelet-derived growth factor-induced translocation and cell migration

The phosphatidylinositol 3-kinase (PI3K) signaling pathway(s) is activated by a variety of agonists to regulate cell migration. Here, we show that the stimulation of mouse embryonic fibroblasts with platelet-derived growth factor (PDGF) induces migration in a PI3K-dependent manner. Cells lacking Akt1/PKBalpha exhibit impaired migration and peripheral ruffling in response to PDGF stimulation, whereas cells lacking Akt2/PKBbeta are normal. In addition, over-expression of Akt1/PKBalpha but not Akt2/PKBbeta is sufficient to restore PDGF-induced cell migration in an Akt1/PKBalpha and Akt2/PKBbeta deficient background. In response to PDGF stimulation, Akt1/PKBalpha selectively translocates to membrane ruffles, however, this localization is abrogated by substituting the linker region of Akt2/PKBbeta. Similarly, expression of an Akt2/PKBalpha chimera containing the linker region of Akt1/PKBalpha restored PDGF-induced migration in cells lacking both Akt1/PKBalpha and Akt2/PKBbeta. Finally, over-expression of constitutively active Rac rescues PDGF-induced migration defects in cells lacking Akt1/PKBalpha. Given these results, we suggest that Akt1/PKBalpha controls cell migration by selectively translocating to the leading edge and activating Rac.