Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.     

Large deformation shear properties of liver tissue

Characterization of the mechanical properties of soft biological tissues is important for establishing the mechanical tolerances of the tissues, and for input to computational models. In this work, the viscoelastic properties of bovine liver tissue in shear loading have been measured using relaxation and constant shear rate loading. The tissue is nonlinearly viscoelastic for strains greater than 0.2%, has a yield strain of approximately 10, and shows moderate strain-rate sensitivity. The response can be modelled using a nonlinear viscoelastic differential model previously developed for brain tissue.     

KfoE encodes a fructosyltransferase involved in capsular polysaccharide biosynthesis in Escherichia coli K4

Escherichia coli K4 synthesizes a capsular polysaccharide (CPS) consisting of a fructose-branched chondroitin (GalNAc-GlcA(fructose)n), which is a biosynthetic precursor of chondroitin sulfate. Here, the role of kfoE in the modification of the chondroitin backbone was investigated using knock-out and recombinant complementation experiments. kfoE disruption and complementation had no significant effect on cell growth. CPS production was increased by 15 % in the knock-out strain, and decreased by 21 % in the knock-out strain complemented with recombinant kfoE. CPS extracted from the knock-out strain was chondroitin, whereas CPS extracted from the complemented strain was a fructose-branched chondroitin. The results demonstrated that the kfoE gene product altered the fructose group at the C3 position of the GlcA residue during production of K4CPS.     

Evaluation of aroma active compounds in <Emphasis Type="Italic">Tuber</Emphasis> fruiting bodies by gas chromatography–olfactometry in combination with aroma reconstitution and omission test

The aroma active compounds of three Tuber fruiting bodies (i.e., Tuber himalayense, Tuber indicum, and Tuber sinense) were firstly systematically evaluated by instrumental gas chromatography–olfactometry combining with quantitative analysis, aroma reconstitution, and omission tests. Twelve aroma active compounds were characterized by aroma extract dilution analysis, and 3-(methylthio) propanal, 3-methylbutanal, and 1-octen-3-ol with the highest flavor dilution (FD) factor (i.e., 1,024–2,048) were suggested as key contributors to the aroma. Odor activity value (OAV) was employed to determine the relative contribution of each compound to the aroma, and the compound with the highest FD factor also had the highest OAV (i.e., 10,234–242,951). Then, the synthetic blends of odorants (aroma reconstitution) were prepared with OAV larger than 15, and their aromas were very similar to the originals. Omission tests were carried out to verify the significance of 3-(methylthio) propanal, 1-octen-3-ol, and 3-methylbutanal as key compounds in the aroma of tested Tuber fruiting bodies.     

Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats

OBJECTIVE: The aim of this study was to develop a feasible approach to promote bone healing in osteoporotic rats using autogenous bone tissue-engineering and gene transfection of human bone morphogenetic protein 2 (hBMP-2). METHODS: Bone marrow stromal cells (BMSCs) from the left tibia of osteoporotic rats were transfected with the hBMP-2 gene in vitro which was confirmed by immunohistochemistry, in situ hybridization and Western blotting. Autogenous transfected or untransfected BMSCs were seeded on macroporous coral hydroxyapatite (CHA) scaffolds. Each cell-scaffold construct was implanted into a defect site which was created in the ramus of the mandible of osteoporotic rats. Four or eight weeks after implantation in situ hybridization was performed in BMSCs transfected with hBMP-2, X-ray examinations, histological and histomorphological analyses were used to evaluate the effect of tissue-engineered bone on osseous defect repair. RESULTS: Newly formed bone was observed at the margin of the defect 4 weeks after implantation with BMSCs transfected with BMP-2. Mature bone was observed 8 weeks after treatment. In the control group there was considerably less new bone and some adipose tissue was observed at the defect margins 8 weeks after implantation. CONCLUSIONS: Autogenous cells transfected with hBMP-2 promote bone formation in osteoporotic rats. BMSC-mediated BMP-2 gene therapy used in conjunction with bone tissue engineering may be used to successfully treat bone defects in osteoporotic rats. This method provides a powerful tool for bone regeneration and other tissue engineering.     

Preliminary results on nematicidal activity from culture filtrates of Basidiomycetes against the pine wood nematode,Bursaphelenchus xylophilus (Aphelenchoididae)

One hundred and eighty one fungal species that were isolated from the fresh fruiting bodies collected in the Mountains of Pu Er County of Yunnan Province, China were tested on the pine wood nematode,Bursaphelenchus xylophilus in vitro. Each fungal species was grown in Czapek broth and potato dextrose broth (PDB). Fifteen filtrates fromAmauroderma austrosinense, Amauroderma macer, Filoboletus sp.,Laccaria tortilis, Lactarius gerardii, Lentinula edodes, Oudemansiella longipes, Oudemansiella mucida, Peziza sp.,Pleurotus sp.,Sinotermitomyces carnosus (two strains),Strobilomyces floccopus, Termitomyces albuminosus, Tylopilus striatulus grown on PDB were found to be pathogenic to the tested nematodes. Eleven filtrates fromAmanita junguillea, Amanita sp.,Daedalea sepiaria, Fistulina hepatica, Omphalotus olearius, Oudemansiella mucida, Peziza sp.,Pleurotus pulmatus, Ramaria sp.,Tricholoma conglobatum, Tylopilus striatulus grown on Czapek broth were also pathogenic to the nematodes. When screening for nematicidal potential of fungi, it is important to study the growth medium conditions necessary to obtain the optimal nematicidal effect as fungal filtrates growing on different liquid media showed a very inconsistent toxicity towards nematodes.     

Comparison of three ELISA kits for the differentiation of foot-and-mouth disease virus-infected from vaccinated animals

A study was performed to validate 3 FMDV 3ABC-I-ELISA kits developed in China for the differentiation of FMDV infected and vaccinated animals. Sets of sera from naive and vaccinated cattle as well as from cattle that had been infected were tested for antibodies against nonstructural proteins (NSPs) of FMDV by commercial diagnosis kits, Ceditest® FMDV-NS (Ceditest® kit), UBI® FMDV NONSTRUCTURAL PROTEIN ELISA DIRECTION INSERT (UBI® kit) and a FMDV 3ABC-I-ELISA kit developed at the Lanzhou Veterinary Research Institute. The test parameters (sensitivity and specificity) of the three kits were determined, and the result obtained from FMD 3ABC-I-ELISA kit was compared with that obtained from two foreign kits. The results indicated that the coincidence rate between the FMDV 3ABC-I-ELISA and Ceditest® kits was 98.05%, and the coincidence rate between the FMDV 3ABC-I-ELISA and UBI® kits was 94.4%; the sensitivity of both Ceditest® and FMDV 3ABC-I-ELISA kit was 100%. However, the sensitivity of the UBI® kit was only 81.8%. With sera from naive or vaccinated non-infected animals, the specificity of all tests exceeded 90%.     

Design and fabrication development of a micro flow heated channel with measurements of the inside micro-scale flow and heat transfer process

The current work provides a design and fabrication technique for a micro channel system that can provide a uniform heat flux boundary condition on the channel wall and a well insulation on the wall to prevent heat loss from the channel to the outside ambient. Therefore, detailed micro-scale flow and heat transfer process and information along the channel can be studied. Semiconductor sensor material was selected to fabricate both the heaters and the arrays of temperature sensors on a silicon substrate. These heaters and sensors were then moved to a low thermal conductivity epoxy-glass substrate for fabrication of the channel. Design consideration and fabrication techniques involved in this processes will be discussed. A final measurement for the validation of the heaters and the sensors fabricated and a study of the flow friction behavior and the heat transfer coefficient distributions inside the micro channel will be presented. The local Nusselt number distrubution inside the micro channel is reported the first time in the open literature.     

Winter spatial distribution of fish larvae assemblages relative to the hydrography of the waters surrounding Taiwan

The aim of this study was to investigate the species composition and distribution of fish larvae in relation to hydrographic conditions in the waters surrounding Taiwan Island (TI) in February 2003. In total, 242 kinds of fish larvae belonging to 127 genera and 75 families were recognized. Among these, 109 taxa were identified to the family or genus level, others to the species level. The 12 predominant types, which constituted 71% of the total fish larvae, were Engraulis japonica, Scomber sp., Diaphus spp., Benthosema pterotum, Carangoides ferdau, Embolichthys mitsukurii, Maurolicus sp., unidentified Myctophidae, Gonostoma gracile, Trichiurus lepturus, unidentified Gobiidae, and Myctophum asperum. The distribution of fish larvae showed a clear association with water masses around TI, with higher abundances and lower species richness northwest of TI where the China Coastal Current prevails, and lower abundances and higher species diversity east of TI where the Kuroshio Current dominates. Cluster analysis distinguished three station groups and four species groups, and the distribution patterns of fish larvae also corresponded to hydrographic conditions. The total abundances of fish larvae and eight of the 12 predominant taxa showed significant and positive correlations with zooplankton abundance, which suggests that food source might be a key factor determining the abundance and distribution of fish larvae during the winter.     

Overexpression of fucosyltransferase IV in A431 cell line increases cell proliferation

Fucosyltransferase IV is an essential enzyme that catalyzes the synthesis of fucosylated oligosaccharides by transferring GDP-fucose to the terminal N-acetylglucosamine with the alpha1,3-linkage. Lewis Y oligosaccharide has a terminal alpha1,3-linked fucose residue and elevation of Lewis Y level is seen in many epithelial cancers. The mechanism of Lewis Y elevation in neoplastic cells is still largely unknown. To study the impact of fucosyltransferase IV on Lewis Y expression and its role on neoplastic cell proliferation, a pEGFP-N1-FUT4 recombinant plasmid was developed and stably transfected into A431 cells. We found that fucosyltransferase IV overexpression promoted cell proliferation and increased the expression of proliferating cell nuclear antigen that correlated with Lewis Y augmentation. Cell cycle analysis demonstrated that fucosyltransferase IV overexpression facilitated cell cycle progression. In conclusion, fucosyltransferase IV overexpression augments Lewis Y expression to trigger neoplastic cell proliferation. These studies suggest that fucosyltransferase IV may serve as a potential therapeutic target for the treatment of Lewis Y-positive epithelial cancers.