Amino acid requirements for maturation of rhesus monkey (Macacca mulatta) oocytes in culture

This study evaluated the effects of different amino acid formulations on supporting meiotic and cytoplasmic maturation of rhesus monkey (Macacca mulatta) oocytes in vitro. Five hundred and forty-six cumulus-oocyte complexes (COCs) aspirated from unstimulated adult monkey follicles (greater than or equal to 1000 mum in diameter) were cultured in either modified Connaught Medical Research Laboratories 1066 medium (mCMRL-1066) or in one of eight chemically defined media (modified basic medium 5 supplemented with 5.5 mmol glucose l(-1), 0.003 mmol pantothenic acid l(-1) and different amino acid formulations) as below: (1) modified basic medium 5 (mBM5) containing no amino acid; (2) mBM5 + 0.2 mmol glutamine l(-1); (3) mBM5 + 11 amino acids from hamster embryo culture medium 6 (HECM-6) (11 AA); (4) mBM5 + Eagle's non-essential amino acids (NEA); (5) mBM5 + NEA + 0.2 mmol glutamine l(-1); (6) mBM5 + Eagle's essential amino acids (EA) without glutamine; (7) mBM5 + EA + 0.2 mmol glutamine l(-1); (8) mBM5 + Eagle's 20 amino acids (20 AA) + 0.2 mmol glutamine l(-1); and (9) mCMRL-1066 (control). All media contained FSH, LH, oestradiol and progesterone. After maturation, mature oocytes were subjected to the same fertilization and embryo culture procedures. COCs matured in treatment 5 had greater potential to progress to metaphase II (66%; P < 0.05) than did those in treatments 1 (37.3%), 2 (48.3%)f 3 (41%), 6 (41%) and 9 (43%). Oocytes matured in treatment 8 had the best morula (53%) and blastocyst (18%) developmental responses (P<0.05). The lowest (P<0.05) morula and blastocyst developmental responses were obtained from COCs matured in treatments 1 (0%) and 6 (8%). The other media supported intermediate embryonic development (range 11-38% of morula and blastocyst). These results indicate that the choice of amino acids affects the competence of oocyte maturation and that Eagle's 20 AA with 0.2 mmol glutamine l(-1) is more efficient than the other amino acid formulations for maturation of rhesus monkey oocytes.

Biodegradable Interpolyelectrolyte Complexes Based on Methoxy Poly(ethylene glycol)-b-poly(alpha,L-glutamic acid) and Chitosan

We synthesized methoxy poly(ethylene glycol)-b-poly(alpha,L-glutamic acid) (mPEGGA) diblock copolymer by ring-opening polymerization of N-carboxy anhydride of gamma-benzyl-L-glutamate (NCA) using amino-terminated methoxy polyethylene glycol (mPEG) as macroinitiator. Polyelectrolyte complexation between mPEGGA as neutral-block-polyanion and chitosan (CS) as polycation has been scrutinized in aqueous solution as well as in the solid state.

Novel temperature- and pH-responsive graft copolymers composed of poly(L-glutamic acid) and poly(N-isopropylacrylamide)

A series of novel temperature- and pH-responsive graft copolymers, poly(L-glutamic acid)-g-poly(N-isopropylacrylamide), were synthesized by coupling amino-semitelechelic poly(N-isopropylacrylamide) with N-hydroxysuccinimide-activated poly(L-glutamic acid). The graft copolymers and their precursors were characterized, by ESI-FTICR Mass Spectrum, intrinsic viscosity measurements and proton nuclear magnetic resonance (H-1 NMR). The phase-transition and aggregation behaviors of the graft copolymers in aqueous solutions were investigated by the turbidity measurements and dynamic laser scattering.

Structural diversity of lanthanide-amino acid complexes under near physiological pH conditions and their recognition of single-stranded DNA

We reported here four structures of lanthanide-amino acid complexes obtained under near physiological pH conditions and their individual formula can be described as [Tb-2(DL-Cys)(4)(H2O)(8)]Cl-2 (1), [Eu-4(mu(3)-OH)(4)(L-Asp)(2)(L-HAsp)(3)(H2O)(7)] Cl center dot 11.5H(2)O (2), [Eu-8-(L-HVal) (16)(H2O)(32)]Cl-24 center dot 12.5H(2)O (3), and [Tb-2(DL-HVal)(4)(H2O)(8)]Cl-6 center dot 2H(2)O (4). These complexes showed diverse structures and have shown potential application in DNA detection. We studied the interactions of the complexes with five single-stranded DNA and found different fluorescence enhancement, binding affinity and binding stoichiometry when the complexes are bound to DNA.

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase1. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.

Synthesis of a novel structural triblock copolymer of poly(gamma-benzyl-L-glutamic acid)-b-poly(ethylene oxide)-b-poly(epsilon-caprolactone)

A novel structural triblock copolymer of poly(gamma-benzyl-L-glutamic acid)-b-poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PBLG-PEO-PCL) was synthesized by a new approach in the following three steps: (1) sequential anionic ring opening polymerization (ROP) of ethylene oxide and epsilon-caprolactone with an acetonitrile/potassium naphthalene initiator system to obtain a diblock copolymer CN-PEO-PCL with a cyano end-group; (2) conversion of the CN end-group into NH2 end-group by hydrogenation to obtain NH2-PEO-PCL; (3) ROP of gamma-benzyl-L-glutamate-N-carboxyanhydrides (Bz-L-GluNCA) with NH2-PEO-PCL as macroinitiator to obtain the target triblock copolymer. The structures from CN-PEO precursor to the triblock copolymers were confirmed by FT-IR and H-1 NMR spectroscopy, and their molecular weights were measured by gel permeation chromatography. The monomer of Bz-L-GluNCA can react almost quantitatively with the amino end-groups of NH2-PEO-PCL macroinitiator by ROP.

Reversible B/Z-DNA transition under the low salt condition and non-B-form polydApolydT selectivity by a cubane-like europium-L-aspartic acid complex

We report here that a cubane-like europium-L-aspartic acid complex at physiological pH can discriminate between DNA structures as judged by the comparison of thermal denaturation, binding stoichiometry, temperature-dependent fluorescence enhancement, and circular dichroism and gel electrophoresis studies. This complex can selectively stabilize non-B-form DNA polydApolydT but destabilize polydGdCpolydGdC and polydAdTpolydAdT. Further studies show that this complex can convert B-form polydGdCpolydGdC to Z-form under the low salt condition at physiological temperature 37 degrees C, and the transition is reversible, similar to RNA polymerase, which turns unwound DNA into Z-DNA and converts it back to B-DNA after transcription. The potential uses of a left-handed helix-selective probe in biology are obvious. Z-DNA is a transient structure and does not exist as a stable feature of the double helix. Therefore, probing this transient structure with a metal-amino acid complex under the low salt condition at physiological temperature would provide insights into their transitions in vivo and are of great interest.

Synthesis and characterization of RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) triblock copolymer

Advances in tissue engineering require biofunctional scaffolds that can provide not only physical support for cells but also chemical and biological cues needed in forming functional tissues. To achieve this goal, a novel RGD peptide grafted poly(ethylene glycol)-b-poly(L-lactide)-b-poly(L-glutamic acid) (PEG-PLA-PGL/RGD) was synthesized in four steps (1) to prepare diblock copolymer PEG-PLA-OH and to convert its -OH end group into -NH2 (to obtain PEG-PLA-NH2), (2) to prepare triblock copolymer PEG-PLA-PBGL by ring-opening polymerization of NCA (N-carboxyanhydride) derived from benzyl glutamate with diblock copolymer PEG-PLA-NH2 as macroinitiator, (3) to remove the protective benzyl groups by catalytic hydrogenation of PEGPLA-PBGL to obtain PEG-PLA-PGL, and (4) to react RGD (arginine-glycine-(aspartic amide)) with the carboxyl groups of the PEG-PLA-PGL. The structures of PEG-PLA-PGL/RGD and its precursors were confirmed by H-1 NMR, FT-IR, amino acid analysis, and XPS analysis. Addition of 5 wt % PEG-PLA-PGL/RGD into a PLGA matrix significantly improved the surface wettability of the blend films and the adhesion and proliferation behavior of human chondrocytes and 3T3 cells on the blend films. Therefore, the novel RGD-grafted triblock copolymer is expected to find application in cell or tissue engineering.

PolydA and polyrA self-structured by a europium and amino acid complex

Different DNA selectivity was found for the newly synthesized europium-L-valine complex. Unexpected DNA and RNA selection results showed that europium-L-valine complex can cause single-stranded polydA and polyrA to self-structure. The sigmoidal melting curve profiles indicate the transition is cooperative, similar to the cooperative melting of a duplex DNA. This is different from another europium amino acid complex, europium-L-aspartic acid complex which can induce B-Z transition under the low salt condition. To our knowledge, there is no report to show that a metal-amino acid complex can cause the self-structuring of single-stranded DNA and RNA.

STUDIES ON THE COORDINATION OF TB(III) AND CA(III) WITH AMINO-ACID UNDER THE PHYSIOLOGICAL CONDITION

Tb(Ca)-glycine, Tb(Ca)-alanine, Tb(Ca)-glycine-alanine systems were studied by potentiometry (37%, I = 0.15 mol/L NaCl). The stability constants of complexes and distribution of species in ternary system were obtained. The results show CaTb for the ternary complexes. The species convert each other with changing pH values.

An Amino Acid Substitution Matrix for Protein Conformation Identification

Amino acid substitution matrices play an essential role in protein sequence alignment, a fundamental task in bioinformatics. Most widely used matrices, such as PAM matrices derived from homologous sequences and BLOSUM matrices derived from aligned segments of PROSITE, did not integrate conformation information in their construction. There are a few structure-based matrices, which are derived from limited data of structure alignment. Using databases PDB_SELECT and DSSP, we create a database of sequence-conformation blocks which explicitly represent sequence-structure relationship. Members in a block are identical in conformation and are highly similar in sequence. From this block database, we derive a conformation-specific amino acid substitution matrix CBSM60. The matrix shows an improved performance in conformational segment search and homolog detection.

Effects of Molecular Length, Ortientation and Amino Acid Mutation on Kinetics of Selectin/Ligand interactions

Receptor/ligand interactions are basic issues to cell adhesion, which are important to many physiological and pathological processes such as lymphocyte-mediated cytotoxicity, tumor metastasis and inflammatory reactionl. Selectin/carbohydrate ligand bindings have been found to mediate the fast rolling of leukocytes on activated endothelial monolayer. Kinetic rate and binding affinity constants are essential determinants of cell adhesion...

A single amino acid defines cross-species reactivity of tree shrew (Tupaia belangeri) CD1d to human invariant natural killer T (iNKT) cells

P>The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evoluti