Cell surface biotinylation by azaelectrocyclization: easy-handling and versatile approach for living cell labeling

Versatile method for living cell labeling has been established. Cell surfaces are initially biotinylated by azaelectrocyclization, and then treated with the fluorescence-labeled avidin or the anti-biotin antibody.     

PfbA, a novel plasmin- and fibronectin-binding protein of Streptococcus pneumoniae, contributes to fibronectin-dependent adhesion and antiphagocytosis

Streptococcus pneumoniae is a major causative agent of mortality throughout the world. The initial event in invasive pneumococcal disease is the attachment of pneumococci to epithelial cells in the upper respiratory tract. Several bacterial proteins can bind to host extracellular matrix proteins and function as adhesins and invasins. To identify adhesins or invasins on the pneumococcal cell surface, we searched for several proteins with an LPXTG anchoring motif in the whole-genome sequence of Streptococcus pneumoniae and identified one, which we called PfbA (plasmin- and fibronectin-binding protein A), that bound to human serum proteins. Immunofluorescence microscopy and fluorescence-activated cell sorter analysis revealed that PfbA was expressed on the pneumococcal cell surface. A DeltapfbA mutant strain was only half as competent as the wild-type strain at adhering to and invading lung and laryngeal epithelial cells. In addition, epithelial cells infected with DeltapfbA showed morphological changes, including cell flattening and a loss of microvilli, that did not occur in cells infected with the wild-type strain. The mutant strain also exhibited a weaker antiphagocytotic activity than wild type in human peripheral blood. Moreover, the growth of wild-type bacteria in human whole blood containing anti-PfbA antibodies was reduced by approximately 50% after 3 h compared with its growth without the antibody. These results suggest that PfbA is an important factor in the development of pneumococcal infections.     

Group A streptococcal cysteine protease degrades C3 (C3b) and contributes to evasion of innate immunity

A relative lack of neutrophils around Streptococcus pyogenes is observed in streptococcal toxic shock syndrome (STSS). Because the bacteria spread rapidly into various organs in STSS, we speculated that S. pyogenes is equipped with molecules to evade the host innate immune system. Complement C3b opsonizes the pathogen to facilitate phagocytosis, and a complex of C3b converts C5 into anaphylatoxin. Because we found that C3 (C3b) is degraded in sera from patients with STSS, we investigated the mechanism of C3 (C3b) degradation by S. pyogenes. We incubated human C3b or serum with recombinant SpeB (rSpeB), a wild-type S. pyogenes strain isolated from an STSS patient or its isogenic DeltaspeB mutant and examined the supernatant by Western blotting with anti-human C3b. Western blot and Biacore analyses revealed that rSpeB and wild-type S. pyogenes rapidly degrade C3b. Additionally, C3 (C3b) was not detected in sera collected from infected areas of STSS patients. Furthermore, the survival rate in human blood and in mice was lower for the DeltaspeB mutant than the wild-type strain. Histopathological observations demonstrated that neutrophils were recruited to and phagocytosed the DeltaspeB mutant, whereas with the wild-type strain, few neutrophils migrated to the site of infection, and the bacteria spread along the fascia. We observed the degradation of C3 (C3b) in sera from STSS patients and the degradation of C3 (C3b) by rSpeB. This suggests that SpeB contributes to the escape of S. pyogenes from phagocytosis at the site of initial infection, allowing it to invade host tissues during severe infections.     

Stabilization of water-in-oil emulsion by <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 and its application to biotransformation

Rhodococcus opacus B-4, which has recently been isolated as an organic solvent-tolerant bacterium, stabilized water-in-oil (w/o) emulsions by inhibition of droplet coalescence when the cells were dispersed in 90% (v/v) organic solvents. Confocal microscopy revealed that many bacterial cells assembled at the interface between oil and water droplets, though free cells were also detectable at the inside of water droplets. Bacterial cells in the w/o emulsion were capable of utilizing both a water-soluble (glucose) and an oil-soluble substrate (oleic acid) as an energy source. Availability of the w/o emulsion as an immobilized cell system in organic solvents was demonstrated using production of indigo from indole and production of o-cresol from toluene as model conversions. When glucose and oleic acid were simultaneously supplied as energy sources, the w/o emulsion culture of R. opacus B-4 produced indigo and o-cresol at levels of 0.217 and 2.12 mg ml⁻¹, respectively, by 12 h.     

Cisplatin differently affects amino terminal and carboxyl terminal domains of HSP90

The 90-kDa heat shock protein (HSP90) is a molecular chaperone that assists in the folding and assembly of proteins in the cytosol. We previously demonstrated that the antineoplastic reagent, cisplatin, inhibits the aggregation prevention activity of mammalian HSP90. We now show that cisplatin binds both the amino terminal and carboxyl terminal domains of the human HSP90 and differently affects these two domains. Cisplatin blocks the aggregation prevention activity of HSP90C, but not HSP90N. In contrast, cisplatin induces a conformational change in HSP90N, but not HSP90C. These results indicate that cisplatin modulates the HSP90 activities through two different mechanisms using the two distinct binding sites of the HSP90 molecule.     

A strong exonic splicing enhancer in dystrophin exon 19 achieve proper splicing without an upstream polypyrimidine tract

Proper splicing is known to proceed under the control of conserved cis-elements located at exon-intron boundaries. Recently, it was shown that additional elements, such as exonic splicing enhancers (ESEs), are essential for the proper splicing of certain exons, in addition to the splice donor and acceptor site sequences; however, the relationship between these cis-elements is still unclear. In this report, we utilize dystrophin exon 19 to analyse the relationship between the ESE and its upstream acceptor site sequences. Dystrophin exon 19, which maintains adequate splicing donor and acceptor consensus sequences, encodes exonic splicing enhancer (dys-ESE19) sequences. Splice pattern analysis, using a minigene reporter expressed in HeLa cells, showed that either a strong polypyrimidine tract (PPT) or a fully active dys-ESE19 is sufficient for proper splicing. Each of these two cis-elements has enough activity for proper exon 19 splicing suggesting that the PPT, which is believed to be an essential cis-element for splicing, is dispensable when the downstream exon contains a strong ESE. This compensation was only seen in living cells but not in 'in vitro splicing'. This suggests the possibility that the previous splicing experiments using an in vitro splicing system could underestimate the activity of ESEs.     

BMPs promote proliferation and migration of endothelial cells via stimulation of VEGF-A/VEGFR2 and angiopoietin-1/Tie2 signalling

The differentiation, growth, and survival of endothelial cells (ECs) are regulated by multiple signalling pathways, such as vascular endothelial growth factors (VEGFs) and angiopoietins through their receptor tyrosine kinases, VEGF receptor (VEGFR) 2 and Tie2, respectively. Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-beta family, have been implicated in the development and maintenance of vascular systems. However, their effects on EC proliferation remain to be elucidated. In the present study, we show that BMPs induce the proliferation and migration of mouse embryonic stem cell (ESC)-derived endothelial cells (MESECs) and human microvascular endothelial cells (HMECs). Addition of BMP-4 to culture induced significant proliferation and migration of both types of ECs. BMP-4 also increased the expression and phosphorylation of VEGFR2 and Tie2. These findings suggest that BMP signalling activates endothelium via activation of VEGF/VEGFR2 and Angiopoietin/Tie2 signalling.     

Isolation of apoptosis- and differentiation-inducing substances toward human promyelocytic leukemia HL-60 cells from leaves of Juniperus taxifolia

A chloroform extract of the leaves of Juniperas taxifolia exhibited a marked antiproliferative effect on human promyelocytic leukemia HL-60 cells at a concentration of 2.5 microg/ml. Deoxypodophyllotoxin (4) was identified in the extract as an outstanding antiproliferative compound, and five diterpenes (1-3, 5, and 6) were isolated as known compounds with weak or no cytotoxicity. These compounds were examined for their respective apoptosis- and differentiation-inducing activities toward HL-60 cells by DNA fragmentation and NBT-reducing assays, respectively. Among them, 7alpha-hydroxysandaracopimaric acid (6) was found to have a potent differentiation-inducing activity in a dose-dependent manner at 0.125-2 microg/ml (0.39-6.29 microM), together with apoptosis-inducing activity at concentrations of more than 2.5 microg/ml (7.86 microM). Deoxypodophyllotoxin (4) that exerted cytotoxic and apoptosis-inducing activities at 2 ng/ml (5 nM) did not induce differentiation at the same concentration, and the other diterpenes (1-3 and 5) showed no effect on cell differentiation, even at 5 microg/ml. It was thus demonstrated for the first time that 7alpha-hydroxysandaracopimaric acid was an effective differentiation-inducing compound toward HL-60 cells.     

Protofibril assemblies of the arctic, Dutch, and Flemish mutants of the Alzheimer's Abeta1-40 peptide

Using a coarse-grained model of the Aβ peptide, we analyze the Arctic (E22G), Dutch (E22Q), and Flemish (A21G) familial Alzheimer's disease (FAD) mutants for any changes in the stability of amyloid assemblies with respect to the wild-type (WT) sequence. Based on a structural reference state of two protofilaments aligned to create the “agitated” protofibril as determined by solid-state NMR, we determine free energy trends for Aβ assemblies for the WT and FAD familial sequences. We find that the structural characteristics and oligomer size of the critical nucleus vary dramatically among the hereditary mutants. The Arctic mutant's disorder in the turn region introduces new stabilizing interactions that better align the two protofilaments, yielding a well-defined protofibril axis at relatively small oligomer sizes with respect to WT. By contrast, the critical nucleus for the Flemish mutant is beyond the 20 chains characterized in this study, thereby showing a strong shift in the equilibrium toward monomers with respect to larger protofibril assemblies. The Dutch mutant forms more ordered protofilaments than WT, but exhibits greater disorder in protofibril structure that includes an alternative polymorph of the WT fibril. An important conclusion of this work is that the Dutch mutant does not support the agitated protofibril assembly. We discuss the implications of the structural ensembles and free energy profiles for the FAD mutants in regards to interpretation of the kinetics of fibril assembly using chromatography and dye-binding experiments.