A heavily glycosylated soluble protein was purified using a combination of lectin affinity and size exclusion chromatography from a soluble extract of uncalcified dorsal cuticle of blue crab ...Callinectes sapidus removed at ecdysis. Similarities in apparent molecular mass and carbohydrate composition suggest that this protein is the same species previously shown to disappear from soluble extracts coincidentally with the onset of mineral deposition in the newly exposed post-molt cuticle. The amino acid sequence of the N-terminal portion of the core polypeptide was determined and polyclonal antibodies were raised against both the purified glycoprotein and the peptide. Immunoblots of unfractionated soluble extracts taken at various times post-molt illustrated that the anti-peptide antibody recognized several polypeptides with electrophoretic mobilities that differ from the purified glycoprotein. These bands may be deglycosylation products which would not have been purified due to different lectin affinity or size. Immunohistochemical analysis indicated uniform protein distribution in the exocuticle at ecdysis, but decreased antibody binding at the interprismatic septa by 2 h post-molt. The location of the protein is therefore the negative image of the calcification pattern in the exocuticle and provides a spatial pattern to correlate with the previously reported temporal events. This strengthens the hypothesis that the glycoprotein under investigation is an inhibitor of calcite nucleation or of initial amorphous calcium carbonate accumulation.
The structure of phycoviolobilin, the photoactive chromophore of α-phycoerythrocyanin, is incompatible with a chromophore ligation to the apoprotein via SH-addition (cysteine) to a Δ3,3
1-double bond ...of the phycobilin. The two putative phycoerythrocyanin lyase genes of
Mastigocladus laminosus,
pecE and
pecF, were overexpressed in
Escherichia coli. Their action has been studied on the addition reaction of phycocyanobilin to apo-α-phycoerythrocyanin (PecA). In the absence of the components of α-PEC-phycoviolobilin lyase PecE and PecF, or in the presence of only one of them, phycocyanobilin binds covalently to PecA forming a fluorescent chromoprotein with a red-shifted absorption (
λ
max=641 nm) and low photoactivity (<10%). In the presence of both PecE and PecF, a chromoprotein forms which by its absorption (
λ
max=565 nm) and high photoreversible photochromism (100% type I) has been identified as integral α-phycoerythrocyanin. We conclude that PecE and PecF jointly catalyze not only the addition of phycocyanobilin to PecA, but also its isomerization to the native phycoviolobilin chromophore.
Sheet forming fundamentals are thoroughly addressed in this comprehensive reference for the practical and efficient use of sheet forming technologies. The principle variables of sheet forming ...including the interactions between variables are clearly explained, as a basic foundation for the most effective use of computer aided modeling in process and die design. Topics include stress analysis, formability criteria, tooling, and materials for sheet forming. The book also covers the latest developments in sheet metal forming technology including servo-drive presses and their applications, and advanced cushion systems in mechanical and hydraulic presses.
The protective antioxidant role of idebenone both as free drug and drug-loaded Tween 80-coated polyethyl-2-cyanoacrylate (PECA) nanocapsules is reported. The relationship between oxidative damage and ...apoptotic or nonapoptotic cell death is evaluated in vitro.
Idebenone-loaded nanocapsules were prepared with the interfacial polymerization method in the presence of Tween 80. Human nonimmortalized fibroblasts. under different stress conditions, either 0.5 mM diethylmaleate (DEM) for 60 min or 0.1 mM H2O2 for 30 min, were used as the experimental in vitro model. The production of reactive oxygen species, the cell viability, and the nuclear DNA damage were evaluated. The presence of apoptotic damage was evaluated both by the determination of caspase-3-like protein activity and by Promega's fluorescent apoptotic detection system.
DEM and H2O2 affected the cultured cells in different ways. DEM induced a moderate cellular insult, which was efficaciously antagonized by idebenone-loaded PECA nanocapsules. H2O2 elicited severe damage to nuclear DNA, which was reduced by idebenoneloaded PECA nanocapsules. The free drug was less effective than idebenone-loaded nanocapsules.
The findings reported here demonstrate that an improved antioxidant effect was obtained with a low idebenone concentration (0.5 microM) when the drug was entrapped within Tween 80-coated PECA nanocapsules.
Poly(ethylcyanoacrylate) (PECA) nanocapsules suitable for use as drug delivery systems were prepared by in situ polymerization, adding the monomer to an organic phase and subsequent mixing of the ...latter to an aqueous phase containing a nonionic surfactant. Different preparation conditions have been able to influence the final PECA nanocapsule colloidal suspension. In particular, the kind of organic solvent caused the formation of either simple PECA nanocapsule suspensions (aprotic fully water-miscible solvents) or PECA nanoparticle colloidal suspensions consisting of nanospheres and nanocapsules (protic water-miscible solvent). Both mechanisms, the interfacial precipitation of a pre-formed polymer and the interfacial polymerization, could play a significant role in nanocapsule formation. Also other variables, such as the kind of the nonionic surfactant and the monomer concentration, affected in different ways the nanocapsule formation process.
By PCR method, apo-phycoerythrocyanin alpha -subunit gene (pecA) of Mastigocladus laminosus (M. laminosus) was amplified from its genomic DNA, and then cloned in pBluescript. The pecA gene was ...subcloned into the expression vector pGEMD, and then transformed into E. coli BL21 (DE3). After induction, a new protein of molecular weight 19 x 10 super(3) existing in inclusion body was overexpressed. The expressed product was confirmed to be apo-phycoerythrocyanin alpha -subunit by Dot-ELISA.
Pectinases produced by
Aspergillus flavus and
A. parasiticus are believed to play a significant role in the ability of these fungi to spread in cotton bolls and other crops. Utilizing a DNA probe, ...generated by PCR, of the
Aspergillus niger pgaII gene, we have isolated a novel, constitutively expressed polygalacturonase (PG)-encoding gene (
pecA) from an
A. parasiticus cDNA library. DNA sequence analysis and the deduced amino acid (aa) sequence of
pecA demonstrated significant identity at the nucleotide and aa levels with other PG of fungal origin. Northern blot analysis of RNA isolated from
A. parasiticus grown on either glucose or pectin as the sole carbon source showed that
pecA was expressed during growth in both media.
Captain Michael Peca of the Buffalo Sabres is profiled. Neutralizing the best player of the opposing team becomes an interesting subplot every time the hockey puck is dropped.
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