Poly(methylene co-guanidine) coated alginate as an encapsulation matrix for urease

Urease was encapsulated within alginate beads, coated with poly(methylene co-guanidine) membranes via polyelectrolyte complexation. Membrane thickness increased with reaction time to 53 μm after 80 min, and to 59 μm with an increase in co-guanidine concentration from 2.5 to 20 mg ml⁻¹. A 70% mass and 31% activity yield of urease resulted following encapsulation. Although co-guanidine strongly inhibited freely soluble urease (I_0.5=5.8 μg ml⁻¹ co-guanidine), immobilization stabilized the enzyme against inactivation. Encapsulated activity declined as the polycation concentration used for membrane formation increased; however an activity loss of only 35% was observed when the co-guanidine concentration was as high as 5 mg ml⁻¹. Glucose protected against inactivation, with 0.5 increasing to 28.5 μg ml⁻¹ for the freely soluble enzyme. When the beads were coated with co-guanidine in the presence of glucose, encapsulated urease activity was fully retained.     

Reverse phase liquid chromatography of beta-endorphins and related peptides

Anomalous band broadening of beta-endorphin related polypeptides chromatographed on hydrophobic high performance stationary phases can be attributed to ligand induced conformational changes associated with polypeptide folding. In the presence of anionic lipids the size exclusion chromatographic behaviour of members of the beta-endorphin family also exhibits similar behaviour. These structure-retention and band broadening behaviour of these polypeptides were in accord with predictions made by hydropathy algorithms and amphipathic helix representations. These observations on surface accessibility of key amino acid residues and their interaction as a conformationally induced domains with stationary phase ligands are equally relevant to other peptidic solutes and neurotransmitters.     

Early implantation stages in the marmoset monkey (Callithrix jacchus)

The morphology of the initial stages of implantation in the marmoset monkey (Callithrix jacchus) was studied by obtaining embryos and associated endometrium at timed intervals after ovulation. Estrus cycles were detected by measuring daily levels of plasma progesterone. Following a short follicular phase, circulating levels of progesterone above 20 ng/ml were taken as representing day 1 after ovulation. On this basis, single, twin, and triplet embryos were recovered from six perfused-fixed females on days 13, 16, 19, 23, and 29 after ovulation and prepared in resin for light microscopy. Early implantation stages, 13 and 16 days after ovulation, were characterized by the intrusion of syncytial trophoblast between epithelial cells of the endometrium with minimal cellular damage. Some hyperplasia of epithelium at the margin of the implantation site was evident. The consolidation of the initial attachment was achieved by an increase in syncytial trophoblast underlying the inner cell mass of the embryo which rapidly surrounded and breached maternal capillaries. Although initially separate, the chorions of twin or triplet embryos started to fuse by day 19 after ovulation. This process was complete by day 29 such that embryos shared a common uterine exocoelom surrounded by continuous trophoblast. It was concluded that implantation in the marmoset monkey commenced on days 11-12.5 after ovulation and involved an intrusive mechanism. Although trophoblast penetration of endometrium was superficial, maternal capillaries were tapped at an early stage of implantation. The fusion of chorions of twins and triplets first occurred around day 19 after ovulation.     

Selectively Modulating Conformational States of USP7 Catalytic Domain for Activation

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Isolation of blood-brain barrier-crossing antibodies from a phage display library by competitive elution and their ability to penetrate the central nervous system

The blood-brain barrier (BBB) is a formidable obstacle for delivery of biologic therapeutics to central nervous system (CNS) targets. Whilst the BBB prevents passage of the vast majority of molecules, it also selectively transports a wide variety of molecules required to maintain brain homeostasis. Receptor-mediated transcytosis is one example of a macromolecule transport system that is employed by cells of the BBB to supply essential proteins to the brain and which can be utilized to deliver biologic payloads, such as antibodies, across the BBB. In this study, we performed phage display selections on the mouse brain endothelial cell line, bEND.3, to enrich for antibody single-chain variable fragments (scFvs) that could compete for binding with a known BBB-crossing antibody fragment, FC5. A number of these scFvs were converted to IgGs and characterized for their ability to bind to mouse, rat and human brain endothelial cells, and subsequent ability to transport across the BBB. We demonstrated that these newly identified BBB-targeting IgGs had increased brain exposure when delivered peripherally in mice and were also able to transport a biologically active molecule, interleukin-1 receptor antagonist (IL-1RA), into the CNS. The antagonism of the interleukin-1 system within the CNS can result in the relief of neuropathic pain. We demonstrated that the BBB-targeting IgGs were able to elicit an analgesic response in a mouse model of nerve ligation-induced hypersensitivity when fused to IL-1RA.     

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.)

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23000–28000 and isoelectric points 10.3–10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-d-glucosamine oligosaccharide (GlcNAc)₃. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8–100% acetylated chitosans and (GlcNAc)_4–6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)_2–6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (Ab_PLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.Mention of a trademark, warranty, propriety, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations Ab antibody - BCLVC basic chitinase/lysozyme cv. Valencia callus - BCVC basic chitinase cv. Valencia callus - CE capillary electrophoresis - CM-chitin-RBV carboxymethyl-chitin-remazol brilliant violet - GlcNAc N-acetyl-d-glucosamine - HEWL hen egg-white lysozyme - M_r relativemolecular mass - pI isoelectric point - PLC potato leaf chitinase - PR pathogenesis-related - SEC size exclusion chromatography We thank Mr. M. Burkhart, Ms. T.-T. Ho, and Ms. M. Doherty for their valuable technical assistance. A portion of the funding for this work was made available from the Citrus Production Research Marketing Order by the Division of Marketing and Development, Florida Department of Agriculture and Consumer Services, Bob Crawford, Commissioner.     

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al³⁺, Ca²⁺, Fe³⁺, and Yb³⁺ and the borderline metal ion Cu²⁺, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M ⁿ +-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe³⁺-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe³⁺-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M ⁿ +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu²⁺-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.Abbreviations IDA iminodiacetic acid - IDA-Mⁿ⁺ iminodiacetic acid chelated to metal ion - IMAC immobilized metal affinity chromatography - DHCC dog heart cytochrome c - HHCC horse heart cytochrome c, THCC, tuna heart cytochrome c - HMYO horse skeletal muscle myoglobin - SMYO sheep skeletal muscle myoglobin - HEWL hen egg white lysozyme     

Conformational analysis of neuropeptide Y-[18-36] analogs in hydrophobic environments.

The interactive and conformational behavior of a series of neuropeptide Y-[18-36] (NPY-[18-36]) analogs in hydrophobic environments have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC) and circular dichroism (CD) spectroscopy. The peptides studied comprised a series of 16 analogs of NPY-[18-36], each containing a single D-amino acid substitution. The influence of these single L-->D substitutions on the alpha-helical conformation of the NPY-[18-36] analogs in different solvent environments was determined by CD spectroscopy. Retention parameters related to the hydrophobic contact area and the affinity of interaction were determined with an n-octadecyl (C18) adsorbent. Structural transitions for all peptides were manifested as significant changes in the hydrophobic binding domain and surface affinity between 4 degrees C and 37 degrees C. The results indicated that the central region of NPY-[18-36] (residues 23-33) is important for maintenance of the alpha-helical conformation. Moreover, L-->D amino acid residue substitutions within the N- and C-terminal regions, as well as Asn29 and Leu30, do not appear to affect the secondary structure of the peptide. These studies demonstrate that RP-HPLC provides a powerful adjunct for investigations into the induction of stabilized secondary structure in peptides upon their interaction with hydrophobic surfaces.