Intravitreal delivery of human NgR-Fc decoy protein regenerates axons after optic nerve crush and protects ganglion cells in glaucoma models.

Purpose: Glaucoma is a major cause of vision loss due to retinal ganglion cell (RGC) degeneration. Therapeutic intervention controls increased IOP, but neuroprotection is unavailable. NogoReceptor1 (NgR1) limits adult central nervous system (CNS) axonal sprouting and regeneration.

Human NgR-Fc decoy protein via lumbar intrathecal bolus administration enhances recovery from rat spinal cord contusion.

Axonal growth and neurological recovery after traumatic spinal cord injury (SCI) is limited by the presence of inhibitory proteins in myelin, several of which act via the NgR1 protein in neurons. A truncated soluble ligand-binding fragment of NgR1 serves as a decoy and promotes recovery in acute and chronic rodent SCI models. To develop the translational potential of these observations, we created a human sequence-derived NgR1(310)-Fc protein.

Resonance assignments for Oncostatin M, a 24-kDa alpha-helical protein.

Oncostatin M (OM) is a cytokine that shares a structural and functional relationship with interleukin-6, leukemia inhibitory factor, and granulocyte-colony stimulating factor, which regulate the proliferation and differentiation of a variety of cell types. A mutant version of human OM in which two N-linked glycosylation sites and an unpaired cysteine have been mutated to alanine (N76A/C81A/N193A) has been expressed and shown to be active. The triple mutant has been doubly isotope-labeled with 13C and 15N in order to utilize heteronuclear multidimensional NMR techniques for structure determination.

The high-resolution, three-dimensional solution structure of human interleukin-4 determined by multidimensional heteronuclear magnetic resonance spectroscopy.

The high-resolution three-dimensional solution structure of recombinant human interleukin-4 (IL-4), a protein of approximately 15 kDa which plays a key role in the regulation of B and T lymphocytes, has been determined using three- and four-dimensional heteronuclear NMR spectroscopy. The structure is based on a total of 2973 experimental NMR restraints, comprising 2515 approximate interproton distance restraints, 102 distance restraints for 51 backbone hydrogen bonds, and 356 torsion angle restraints. A total of 30 structures was calculated by means of hybrid distance geometry-simulated annealing, and the atomic rms distribution about the mean coordinate positions for residues 8-129 is 0.

Partial purification of the rat erythrocyte ceruloplasmin receptor monitored by an electrophoresis mobility shift assay.

Ceruloplasmin (Cp), the main copper transport glycoprotein found in the blood, delivers its copper to intracellular proteins via a plasma membrane receptor protein. Electrophoresis mobility shift assays (EMSAs) were originally developed to detect DNA-protein or RNA-protein binding. A new EMSA involving protein-protein binding has been developed in order to follow the extraction and purification of the rat erythrocyte Cp receptor.

A GM-CSF/IL-3 fusion protein promotes neutrophil and platelet recovery in sublethally irradiated rhesus monkeys.

The effects of a human GM-CSF/IL-3 fusion protein (PIXY321) were examined in a primate model of rebound hematopoiesis following sublethal irradiation. The results demonstrated an enhanced rate of both neutrophil and platelet regeneration, as well as functional activation of circulating neutrophils.

A tentacle gel simplifies the purification of ceruloplasmin.

The mechanism of how chloroethylamine-treated agarose greatly simplifies the purification of ceruloplasmin (Cp) by preferentially binding this protein from blood plasma has been investigated. Chloroethylamine readily cyclizes to ethylenimine under alkaline conditions which then polymerizes to polyethylenimine (PEI). Ethylenimine and PEI were detected in the reaction mixture used to generate the resin.

Three-dimensional solution structure of human interleukin-4 by multidimensional heteronuclear magnetic resonance spectroscopy.

The three-dimensional solution structure of recombinant human interleukin-4, a protein of 133 residues and 15.4 kilodaltons that plays a key role in the immune and inflammatory systems, has been solved by multidimensional heteronuclear magnetic resonance spectroscopy. The structure is dominated by a left-handed four-helix bundle with an unusual topology comprising two overhand connections.

Determination of the secondary structure and folding topology of human interleukin-4 using three-dimensional heteronuclear magnetic resonance spectroscopy.

The secondary structure of human recombinant interleukin-4 (IL-4) has been investigated by three-dimensional (3D) 15N- and 13C-edited nuclear Overhauser (NOE) spectroscopy on the basis of the 1H, 15N, and 13C assignments presented in the preceding paper [Powers, R., Garrett, D. S.

1H, 15N, 13C, and 13CO assignments of human interleukin-4 using three-dimensional double- and triple-resonance heteronuclear magnetic resonance spectroscopy.

The assignment of the 1H, 15N, 13CO, and 13C resonances of recombinant human interleukin-4 (IL-4), a protein of 133 residues and molecular mass of 15.4 kDa, is presented based on a series of 11 three-dimensional (3D) double- and triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N/13C-labeled IL-4 with an isotope incorporation of greater than 95% for the protein expressed in yeast.

Thyroid hormone receptor induction by triiodothyronine in tadpole erythrocytes in vivo and in vitro and the effect of cycloheximide and actinomycin-D.

Tadpole erythrocyte nuclei contain specific T3 binding sites which increase in number during spontaneous or T3-induced metamorphosis. In the present studies the increase in the number of T3 binding sites after a T3 injections appeared to be completely prevented by cycloheximide and actinomycin D, inhibitors of protein synthesis and RNA synthesis, respectively. However, in some experiments the effect was not statistically significant.

Isolation, stabilization, and molecular weight estimation of thyroid hormone receptors of tadpole and chick embryo erythrocytes.

Typical procedures for the isolation of triiodothyronine (T3) receptors from mammalian nuclei involve extraction of nuclei with buffers containing divalent cations and 0.40M KCl. However, when applied to tadpole erythrocyte (RBC) nuclei, this method gave low yields of relatively unstable T3 receptors.

Detection of rat ceruloplasmin receptors using fluorescence microscopy and microdensitometry.

Rat ceruloplasmin (rCp) has been labeled with the fluorophores fluorescein and rhodamine by using the isothiocyanate derivatives (FITC, RBITC). High p-phenylenediamine oxidase activity of the resulting conjugates was observed (70-90% of native activity). Polyacrylamide gel electrophoresis showed fluorescein-labeled rCp (FITC-rCp) had an increased mobility, while rhodamine-labeled rCp (RBITC-rCp) showed no increase in mobility when compared to native rCp.

Binding of Cu(II) to non-prosthetic sites in ceruloplasmin and bovine serum albumin.

The binding of Cu(II) to native human, porcine, bovine and ovine ceruloplasmin (Cp) and to bovine serum albumin (bSA) has been studied at pH 7.4, 30 mM barbital buffer. The results were analyzed for the strength and the number of binding sites using Scatchard plots.

Hemolysis of rabbit erythrocytes in the presence of copper ions. Inhibition by albumin and ceruloplasmin.

The hemolysis of red blood cells (RBC) induced by Cu(II) is modified by ceruloplasmin (Cp) and albumin. The time course of hemolysis for rabbit RBC by Cu(II) consisted of two parts, an induction period followed by a catastrophic lysis period. The induction period decreased and the lysis rate increased with increasing Cu(II) concentration.

Progesterone inhibits the uterotrophic effect of relaxin in immature rats.

Injection of progesterone for 3 days before treatment with relaxin inhibited the trophic effect of the peptide in both estrogen-primed and unprimed uteri. The depression in collagen concentration and increase in apparent rate of proline incorporation into collagen induced by relaxin alone were also eliminated, indicating a fundamental blockade of the effect of relaxin in this experimental design as well as a close association of changes in collagen concentration with tissue hypertrophy. The effect of relaxin on incorporation of proline into soluble protein was not blocked by progesterone, however, suggesting a separate mechanism for this anabolic effect of relaxin.

Electron transfer between heme proteins and ceruloplasmin.

Reduced cytochrome-c, reduced myoglobin and oxyhemoglobin respectively have been oxidized to oxidized cytochrome-c, metmyoglobin and methemoglobin by ceruloplasmin. Metmyoglobin and methemoglobin formation was stoichiometric while oxidized cytochrome-c reacted catalytically. Only 50% methemoglobin was formed which suggested that two hemes out of four could transfer electrons.

Structure of a porcine relaxin inactive on the mouse pubic ligament.

In addition to B31 (CM-a) and B28 (CM-B) relaxins, acid-acetone extracts of ovaries of pregnant sows contain a third major relaxin species (relaxin C). The major components of relaxin C possess about half the activity of CM-a or CM-B in the guinea pig palpation assay, but are completely inactive in the mouse pubic ligament assay. Its uterotrophic and protein anabolic effects in ovariectomized, estrogen-primed mice, however, are comparable to those of CM-B.

Triiodothyronine increases translatable albumin messenger RNA in Rana catesbeiana tadpole liver.

The effects of both 3,5,3'-triiodo-L-thyronine and spontaneous metamorphosis on Rana catesbeiana liver mRNA were studied using in vitro translation of isolated liver poly(A)+ RNA in a rabbit reticulocyte lysate system. Conventional phenol extraction methods yielded degraded RNA due to high levels of endogenous ribonucleases released upon homogenization of Rana catesbeiana liver. Isolation of intact total RNA was achieved using the potent ribonuclease denaturant, guanidinium thiocyanate.

A comparative study of copper ion induced lysis of vertebrate red blood cells.

1. Erythrocytes from different vertebrate classes were tested for susceptibility towards copper ion-induced lysis under identical copper ion concentration and per cent cell volumes. 2.

Effects of porcine relaxins upon uterine hypertrophy and protein metabolism in mice.

Two variant forms of porcine relaxin (B and C) are active in producing relaxation of the guinea pig pubic symphysis and in effecting uterine growth in rats. Only relaxin B, however, is active in the mouse pubic ligament assay. These two hormones were compared in mice for their effects upon uterine growth and incorporation of radioactively labeled proline into soluble protein and collagen in vitro and in vivo.

Decrease in triiodothyronine binding sites in chick embryo erythrocytes during early development.

Specific thyroid hormone (TH) binding sites have been detected in nuclei of erythrocytes obtained from developing chick embryos. The binding characteristics and relative affinities for TH analogs were those expected of TH receptors. Nuclear triiodothyronine (T3) saturation analysis was carried out in vitro by incubating intact erythrocytes in M199 medium with 3-200 pM [125I]T3 for 1 hr at 37 degrees C or 20-24 hr at 21 degrees C.

The effect of copper ion on glutathione and hemolysis in rabbit erythrocytes.

The rate of hemolysis and the decline in glutathione (GSH) in rabbit erythrocytes caused by copper (Cu) ions were determined. Prior investigations have proposed that the oxidative stress induced by Cu ion depleted the normal cell protective mechanisms. The decline in GSH has been proposed as a necessary prerequisite for hemolysis.

Perspectives on copper biochemistry.

The biochemistry of the essential trace element copper has been outlined. Following absorption, Cu(II) is transported by serum albumin and transcuprein to the liver where it is incorporated into the plasma Cu-protein, ceruloplasmin, or, possibly, stored as Cu-metallothionein or as superoxide dismutase. Ceruloplasmin is the long-term copper transporter and carries Cu(II) to the tissues for the biosynthesis of key Cu(II) enzymes, especially cytochrome c oxidase, lysyl oxidase and others.

Inhibition of postpartum uterine involution in the rat by relaxin.

The possible contribution of relaxin to the support of uterine accommodation during late gestation by retarding tissue lysis was examined using the involuting postpartum uteri of unilaterally pregnant rats. In otherwise intact animals, twice-daily administration of 0.1 mg of relaxin (porcine fraction B) significantly retarded the regression of both gravid and, to a greater extent, nongravid tissue during the first 4 days postpartum, and collagenolysis was similarly delayed.