In situ photopolymerization of a polymerizable poly(ethylene glycol)-covered phospholipid monolayer on a methacryloyl-terminated substrate

We have prepared a chemically anchored monolayer of PEG (poly(ethylene glycol)) and phospholipid mixture (PEG/phospholipid) on a methacryloyl-terminated substrate by in situ photopolymerization. Both monoacryloyl phospholipid (acryloyl-PC, 1-palmitoyl-2-[12-(acryloyloxy)dodecanoyl]-sn-glycero-3-phosphocholine) and monoacryloyl PEG (acryloyl-PEG, 12-(acryloyloxy)dodecanoyl-PEG) were synthesized by modifyingphospholipid and PEGwith 12-(acryloyloxy)-1-dodecanoic acid and 12-(acryloyloxy)-1-dodecanol, respectively. The surface pressure-area (pi-A) isotherm showed that acryloyl-PEG molecules were stable in the phospholipid monolayer and that they could be evenly inserted into a phospholipid monolayer at the air/water interface. By adding 10 mol % acryloyl-PEG into phosholipid vesicles, we could produce a PEG/phosholipid monolayer on methacryloyl-terminated substrates using vesicle fusion for 3 h. Then, this polymerizable PEG/phospholipid monolayer was in situ photopolymerized onto a methacryloyl-terminated substrate with eosin Y/triethanolamine as co-initiators. Optimal vesicle fusion and irradiation condition were determined with respect to the vesicle fusion time and duration of irradiation. As confirmed by atomic force microscopy and X-ray reflectivity studies, the polymerized PEG/phosholipid surface formed a PEG-covered phospholipid monolayer with thicknesses of 3 and 6 nm for the base phospholipid monolayer and the covering PEG layer, respectively. The chemical anchoring efficiency ofpolymerized PEG and phospholipid molecules, which was calculated by the relative carbon ratio of each surface before and after methanol washing using X-ray photoelectron spectroscopy, was 98%. This polymerized PEG/phosholipid monolayer showed good stability in organic solution due to firm chemical anchoring to a solid surface.     

bPAK-interacting exchange factor may regulate actin cytoskeleton through interaction with actin

p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against bPIX. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of bPIX were generated and characterized. N-C6 Mab detected bPIX as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to bPIX. Using C-A3 Mab possible bPIX interaction with actin in PC12 cells was examined. bPIX Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecipitate bPIX. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of bPIX in the cell lysates. These results suggest that bPIX may not interact with soluble actin but with polymerized F-actin and revealed that bPIX constitutes a functional complex with actin. These data indicate real usefulness of the bPIX Mab in the study of bPIX role(s) in regulation of actin cyoskeleton.     

Synthesis of (±)‐Kempa‐6,8‐dien‐3‐ol (=(2aRS,3SR,4aSR,7RS,7aSR,10bSR,10cSR)‐2,2a,3,4,4a,5,6,7,7a,8,10b,10c‐Dodecahydro‐2a,7,10,10c‐tetramethylnaphth[2,18‐cde]azulen‐3‐ol)

The synthesis of kempa‐6,8‐dien‐3β‐ol ( 4a ), as a synthetic leading model of the natural product 4b , was carried out starting from intermediate 12 , the synthetic route of which has been developed previously (Scheme 1). The conversion of 12 to the model compound 4a involved the elaboration of three structure modifications by three processes, Tasks A, B, and C (see Scheme 2). Task A was achieved by epoxy‐ring opening of 41 with Me₃SiCl (Scheme 9), and Task B being performed by oxidation at the 13‐position, followed by hydrogenation, and then epimerization (Schemes 4 and 5). The removal of the 2‐OH group from 12 (Task C) was achieved via 30b according to Scheme 6, whereby 30b was formed exclusively from 30a / 31a 1 : 1 (Scheme 7). In addition, some useful reactions from the synthetic viewpoint were developed during the course of the present experiments.     

Electron beam pretreatment of sewage sludge before anaerobic digestion

The pretreatment of waste-activated sludge (WAS) by electron beam irradiation was studied in order to improve anaerobic sludge digestion. The irradiation dose of the electron beam was varied from 0.5 to 10 kGy. Batch and continuous-flow stirred tank reactors (CFSTRs) were operated to evaluate the effect of the electron beam pretreatment on anaerobic sludge digestion. Approximately 30–52% of the total chemical oxygen demand (COD) content of the WAS was solubilized within 24 h after electron beam irradiation. A large quantity of soluble COD, protein, and carbohydrates leached out from cell ruptures caused by the electron beam irradiation. Volatile fatty acids production from the irradiated sludge was approx 90% higher than that of the unirradiated sludge. The degradation of irradiated sewage sludge was described by two distinct first-order decay rates (k ₁ and k ₂). Most initial decay reaction accelerated within 10 d, with an average k ₁ of 0.06/d for sewage sludge irradiated at all dosages. The mean values for the long-term batch first-order decay coefficient (k ₂) were 0.025/d for irradiated sewage sludge and 0.007/d for unirradiated sludge. Volatile solids removal efficiency of the control reactor fed with unirradiated sewage sludge at a hydraulic retention time (HRT) of 20 d was almost the same as that of the CFSTRs fed with irradiated sludge at an HRT of 10 d. Therefore, disintegration of sewage sludge cells using electron beam pretreatment could reduce the reactor solid retention time by half.     

Improved immunodetection of human papillomavirus E7

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell lines which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.     

Pretreatment of swine wastewater using anaerobic filter

Efforts were made to assess the efficiency of an anaerobic filter packed with porous floating ceramic media and to identify the optimum operational condition of anaerobic filter as a pretreatment of swine wastewater for the subsequent biological removal of nitrogen and phosphorus. A stepwise decrease in hydraulic retention time (HRT) and an increase in organic loading rate (OLR) were utilized in an anaerobic filter reactor at mesophilic temperature (35°C). The optimum operating condition of the anaerobic filter was found to be at an HRT of 1 d. A soluble chemical oxygen demand (COD) removal efficiency of 62% and a total suspended solids removal efficiency of 39% at an HRT of 1 d were achieved with an OLR of 16.0 kg total COD/(m³·d), respectively. The maximum methane production rate approached 1.70 vol of biogas produced per volume of reactor per day at an HRT of 1 d. It was likely that the effluent COD/total Kjeldahl nitrogen ratio, of 22, the COD/total phosphorous ratio of 47, and the high effluent alkalinity >2500 mg/L as CaCO₃ of the anaerobic filter operated at an HRT of 1 d was adequate for the subsequent biological removal of nitrogen and phosphorus.     

Chemoenzymatic Synthesis of Glycopeptides with PglB, a Bacterial Oligosaccharyl Transferase from Campylobacter jejuni

The gram-negative bacterium Campylobacter jejuni has a general N-linked glycosylation pathway encoded by the pgl gene cluster. One of the proteins in this cluster, PglB, is thought to be the oligosaccharyl transferase due to its significant homology to Stt3p, a subunit of the yeast oligosaccharyl transferase complex. PglB has been shown to be involved in catalyzing the transfer of an undecaprenyl-linked heptasaccharide to the asparagine side chain of proteins at the Asn-X-Ser/Thr motif. Using a synthetic disaccharide glycan donor (GalNAc-α1,3-bacillosamine-pyrophosphate-undecaprenyl) and a peptide acceptor substrate (KDFNVSKA), we can observe the oligosaccharyl transferase activity of PglB in vitro. Furthermore, the preparation of additional undecaprenyl-linked glycan variants reveals the ability of PglB to transfer a wide variety of saccharides. With the demonstration of PglB activity in vitro, fundamental questions surrounding the mechanism of N-linked glycosylation can now be addressed.     

Highly sensitive determination of a polymeric hindered amine light stabilizer in polypropylene by reactive thermal desorption-gas chromatography using nitrogen-specific detection

Highly sensitive and specific determination of trace amounts of a polymeric hindered amine light stabilizer (HALS) in polypropylene (PP) materials could be established by improving reactive thermal desorption-gas chromatography (RTD-GC) in the presence of an organic alkali, tetramethylammonium hydroxide. By using nitrogen-phosphorus detection, highly selective detection of the HALS-related components was attained. In addition, the use of a polar poly(ethylene glycol) separation column alleviated the adsorption of minor specific pyrolysis products. This modified RTD-GC method allowed the determination of the polymeric HALS (Mr 1900) in PP even for trace concentrations between 100 and 500 ppm, through observing selectively the characteristic products containing a tetramethylpiperidine moiety, which had been impossible to detect under the previous RTD-GC conditions using a non-polar separation column and conventional flame ionization detection.     

The p38 mitogen-activated protein kinase is involved in stress-induced phospholipase D activation in vascular smooth muscle cells

Oxidative stress has been implicated in mediation of vascular disorders. Earlier study showed that the exposure of vascular smooth muscle cells (VSMC) to pervanadate (hydrogen peroxide plus orthovanadate) resulted in the accumulation of [3H]phosphatidylbutanol. In this study, the effect of pervanadate on the activation of p38 mitogen-activated protein kinase (p38 MAPK) was studied in the VSMC. Pervanadate treatment activated p38 MAPK in a dose-and time-dependent manner. Interestingly, specific inhibition of p38 MAPK with SB203580 attenuated pervanadate-induced PLD activation. This correlates with the finding that expression of dominant negative mutants of MKK3/6 inhibited the PLD activation. SB203580 pretreatment also inhibited other cellular stressors (i.e. high osmolarity and UV light)-induced PLD activation. The possible correlationship of p38 MAPK activation with PKC was examined since PKC is reported to be involved in the pervanadate-induced PLD activation. Calphostin C, a PKC inhibitor, suppressed pervanadate-induced p38 MAPK and PLD activation in a dose-dependent manner. These results suggest that PKC-p38 MAPK may represent an upstream pathway of PLD in the signal transduction of cellular stress.     

Ring‐Opening Reactions of 3‐Aryl‐1‐benzylaziridine‐2‐carboxylates and Application to the Asymmetric Synthesis of an Amphetamine‐Type Compound

Nucleophilic ring‐opening reactions of 3‐aryl‐1‐benzylaziridine‐2‐carboxylates were examined by using O‐nucleophiles and aromatic C‐nucleophiles. The stereospecificity was found to depend on substrates and conditions used. Configuration inversion at C(3) was observed with O‐nucleophiles as a major reaction path in the ring‐opening reactions of aziridines carrying an electron‐poor aromatic moiety, whereas mixtures containing preferentially the syn‐diastereoisomer were generally obtained when electron‐rich aziridines were used (Tables 1–3). In the reactions of electron‐rich aziridines with C‐nucleophiles, S_N2 reactions yielding anti‐type products were observed (Table 4). Reductive ring‐opening reaction by catalytic hydrogenation of (+)‐trans‐(2S,3R)‐3‐(1,3‐benzodioxol‐5‐yl)aziridine‐2‐carboxylate (+)‐trans‐ 3c afforded the corresponding α‐amino acid derivative, which was smoothly transformed into (+)‐tert‐butyl [(1R)‐2‐(1,3‐benzodioxol‐5‐yl)‐1‐methylethyl]carbamate((+)‐ 14 ) with high retention of optical purity (Scheme 6).