PmST3 from <Emphasis Type="Italic">Pasteurella multocida</Emphasis> encoded by <Emphasis Type="Italic">Pm1174</Emphasis> gene is a monofunctional α2–3-sialyltransferase

Pasteurella multocida (Pm) strain Pm70 has three putative sialyltransferase genes including Pm0188, Pm0508, and Pm1174. A Pm0188 gene homolog in Pm strain P-1059 encodes a multifunctional α2–3-sialyltransferase, PmST1, that prefers oligosaccharide acceptors. A Pm0508 gene homolog in the same strain encodes a monofunctional sialyltransferase PmST2 that prefers glycolipid acceptors. Here, we report that the third sialyltransferase from Pm (PmST3) encoded by gene Pm1174 in strain Pm70 is a monofunctional α2–3-sialyltransferase that can use both oligosaccharides and glycolipids as efficient acceptors. Despite the existence of both Pm0188 and Pm0508 gene homologs encoding PmST1 and PmST2, respectively, in Pm strain P-1059, a Pm1174 gene homolog appears to be absent from Pm strains P-1059 and P-934. PmST3 was successfully obtained by cloning and expression using a synthetic gene of Pm1174 with codons optimized for Escherichia coli expression system as the DNA template for polymer chain reactions. Truncation of 35 amino acid residues from the carboxyl terminus was shown to improve the expression of a soluble and active enzyme in E. coli as a C-His₆-tagged fusion protein. This sialidase-free monofunctional α2–3-sialyltransferase is a useful tool for synthesizing sialylated oligosaccharides and glycolipids.     

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS⁺ wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH₂O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log₁₀ in sterile ddH₂O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log₁₀ in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS⁺ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.     

Retinoic acid synthesis and functions in early embryonic development

Retinoic acid (RA) is a morphogen derived from retinol (vitamin A) that plays important roles in cell growth, differentiation, and organogenesis. The production of RA from retinol requires two consecutive enzymatic reactions catalyzed by different sets of dehydrogenases. The retinol is first oxidized into retinal, which is then oxidized into RA. The RA interacts with retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) which then regulate the target gene expression. In this review, we have discussed the metabolism of RA and the important components of RA signaling pathway, and highlighted current understanding of the functions of RA during early embryonic development.     

Antibody repertoires in humanized NOD-scid-IL2Rγ(null) mice and human B cells reveals human-like diversification and tolerance checkpoints in the mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ^null engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγ^null mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential D_H-J_H pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.     

MiR-214 Targets β-Catenin Pathway to Suppress Invasion, Stem-Like Traits and Recurrence of Human Hepatocellular Carcinoma

The down-regulation of miR-214 has previously been observed in human hepatocellular carcinoma (HCC). Here, we demonstrated the down-regulation of miR-214 is associated with cell invasion, stem-like traits and early recurrence of HCC. Firstly, we validated the suppression of miR-214 in human HCC by real-time quantitative RT-PCR (qRT-PCR) in 20 paired tumor and non-tumor liver tissues of HCC patients and 10 histologically normal liver tissues from colorectal cancer patients with liver metastases. Further qRT-PCR analysis of 50 HCC tissues from an independent cohort of HCC patients of whom 29 with early recurrent disease (<2 years) and 21 with late recurrent disease demonstrated that the suppression of miR-214 was significantly more suppressed in samples from HCC patients with early recurrent disease compared those from patients with no recurrence. Re-expression of miR-214 significantly suppressed the growth of HCC cells in vitro and reduced their tumorigenicity in vivo. The enhancer of zeste homologue 2 (EZH2) and β-catenin (CTNNB1) was identified as two potential direct downstream targets of miR-214 through bioinformatics analysis and experimentally validated the miRNA-target interactions with a dual-firefly luciferase reporter assay. In corroborate with this, both EZH2 and CTNNB1 are found to be significantly overexpressed in human HCC biopsies. Since EZH2 can regulate CTNNB1, CTNNB1 can also be an indirect target of miR-214 through EZH2. Silencing EZH2 or CTNNB1 expression suppressed the growth and invasion of HCC cells and induced E-cadherin (CDH1), known to inhibit cell invasion and metastasis. Furthermore, the silencing of miR-214 or overexpression of EZH2 increased EpCAM(+) stem-like cells through the activation of CTNNB1. Interestingly, the up-regulation of EZH2, CTNNB1 and the down-regulation of CDH1 in HCC patients correlated with early recurrent disease and can be an independent predictor of poor survival. Therefore, miR-214 can directly or indirectly target CTNNB1 to modulate the β-catenin signaling pathway in HCC.     

FE65 as a link between VLDLR and APP to regulate their trafficking and processing

Background

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) have been linked to familial Parkinson??s disease, but the underlying pathogenic mechanism remains unclear. We previously reported that loss of PINK1 impairs mitochondrial respiratory activity in mouse brains.

Results

In this study, we investigate how loss of PINK1 impairs mitochondrial respiration using cultured primary fibroblasts and neurons. We found that intact mitochondria in PINK1?/? cells recapitulate the respiratory defect in isolated mitochondria from PINK1?/? mouse brains, suggesting that these PINK1?/? cells are a valid experimental system to study the underlying mechanisms. Enzymatic activities of the electron transport system complexes are normal in PINK1?/? cells, but mitochondrial transmembrane potential is reduced. Interestingly, the opening of the mitochondrial permeability transition pore (mPTP) is increased in PINK1?/? cells, and this genotypic difference between PINK1?/? and control cells is eliminated by agonists or inhibitors of the mPTP. Furthermore, inhibition of mPTP opening rescues the defects in transmembrane potential and respiration in PINK1?/? cells. Consistent with our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1?/? and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. Following FCCP treatment, calcium increases in the cytosol are higher in PINK1?/? compared to wild-type cells, suggesting that intra-mitochondrial calcium concentration is higher in the absence of PINK1.

Conclusions

Our findings show that loss of PINK1 causes selective increases in mPTP opening and mitochondrial calcium, and that the excessive mPTP opening may underlie the mitochondrial functional defects observed in PINK1?/? cells.     

COLONY SIZE OF PHAEOCYSTIS ANTARCTICA (PRYMNESIOPHYCEAE) AS INFLUENCED BY ZOOPLANKTON GRAZERS1

The haptophyte Phaeocystis antarctica G. Karst. is a dominant phytoplankton species in the Ross Sea, Antarctica, and exists as solitary cells and mucilaginous colonies that differ by several orders of magnitude in size. Recent studies with Phaeocystis globosa suggest that colony formation and enlargement are defense mechanisms against small grazers. To test if a similar grazer‐induced morphological response exists in P. antarctica, we conducted incubation experiments during the austral summer using natural P. antarctica and zooplankton assemblages. Dialysis bags that allowed exchange of dissolved chemicals were used to separate P. antarctica and zooplankton during incubations. Geometric mean colony size decreased by 35% in the control, but increased by 30% in the presence of grazers (even without physical contact) over the 15 d incubation. The estimated colonial‐to‐solitary cell carbon ratio was significantly higher in the grazing treatment. These results suggest that P. antarctica colonies would grow larger in the presence of indigenous zooplankton and skew the carbon partitioning significantly toward the colonial phase. While these observations show that the colony size of P. antarctica was affected by a chemical signal related to grazers, the detailed nature and ecological significance of this signal remain unknown.     

Senescence-induced ectopic expression of the A. tumefaciens ipt gene in wheat delays leaf senescence, increases cytokinin content, nitrate influx, and nitrate reductase activity, but does not affect grain yield

The manipulation of cytokinin levels by senescence-regulated expression of the Agrobacterium tumefaciens ipt gene through its control by the Arabidopsis SAG12 (senescence-associated gene 12) promoter is an efficient tool for the prolongation of leaf photosynthetic activity which potentially can affect plant productivity. In the present study, the efficiency of this approach was tested on wheat (Triticum aestivum L.)-a monocarpic plant characterized by a fast switch from vegetative to reproductive growth, and rapid translocation of metabolites from leaves to developing grains after anthesis. When compared with the wild-type (WT) control plants, the SAG12::ipt wheat plants exhibited delayed chlorophyll degradation only when grown under limited nitrogen (N) supply. Ten days after anthesis the content of chlorophyll and bioactive cytokinins of the first (flag) leaf of the transgenic plants was 32% and 65% higher, respectively, than that of the control. There was a progressive increase in nitrate influx and nitrate reductase activity. However, the SAG12::ipt and the WT plants did not show differences in yield-related parameters including number of grains and grain weight. These results suggest that the delay of leaf senescence in wheat also delays the translocation of metabolites from leaves to developing grains, as indicated by higher accumulation of ((15)N-labelled) N in spikes of control compared with transgenic plants prior to anthesis. This delay interferes with the wheat reproductive strategy that is based on a fast programmed translocation of metabolites from the senescing leaves to the reproductive sinks shortly after anthesis.     

H5-type influenza virus hemagglutinin is functionally recognized by the natural killer-activating receptor NKp44

Antiviral immune defenses involve natural killer (NK) cells. We previously showed that the NK-activating receptor NKp44 is involved in the functional recognition of H1-type influenza virus strains by NK cells. In the present study, we investigated the interaction of NKp44 and the hemagglutinin of a primary influenza virus H5N1 isolate. Here we show that recombinant NKp44 recognizes H5-expressing cells and specifically interacts with soluble H5 hemagglutinin. H5-pseudotyped lentiviral particles bind to NK cells expressing NKp44. Following interaction with target cells expressing H5, pseudotyped lentiviral particles, or membrane-associated H5, NK cells show NKp44-mediated induced activity. These findings indicate that NKp44-H5 interactions induce functional NK activation.     

Quantitative multicolor compositional imaging resolves molecular domains in cell-matrix adhesions

Background

Cellular processes occur within dynamic and multi-molecular compartments whose characterization requires analysis at high spatio-temporal resolution. Notable examples for such complexes are cell-matrix adhesion sites, consisting of numerous cytoskeletal and signaling proteins. These adhesions are highly variable in their morphology, dynamics, and apparent function, yet their molecular diversity is poorly defined.

Methodology/Principal Findings

We present here a compositional imaging approach for the analysis and display of multi-component compositions. This methodology is based on microscopy-acquired multicolor data, multi-dimensional clustering of pixels according to their composition similarity and display of the cellular distribution of these composition clusters. We apply this approach for resolving the molecular complexes associated with focal-adhesions, and the time-dependent effects of Rho-kinase inhibition. We show here compositional variations between adhesion sites, as well as ordered variations along the axis of individual focal-adhesions. The multicolor clustering approach also reveals distinct sensitivities of different focal-adhesion-associated complexes to Rho-kinase inhibition.

Conclusions/Significance

Multicolor compositional imaging resolves “molecular signatures” characteristic to focal-adhesions and related structures, as well as sub-domains within these adhesion sites. This analysis enhances the spatial information with additional “contents-resolved” dimensions. We propose that compositional imaging can serve as a powerful tool for studying complex multi-molecular assemblies in cells and for mapping their distribution at sub-micron resolution.