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The effects of temperature and pressure variation on the lyophilization of interleukin-2 (IL-2) were studied. The human recombinant IL-2 used in this study was synthesized in and purified from E. coli, formulated, and then submitted to experimental lyophilization procedures. The collapse temperature of the formulation was first determined to be -25 degrees C by differential scanning calorimetry. The effects of chamber pressure and shelf temperature on IL-2 during lyophilization were evaluated. Lyophilization chamber pressures were varied from 100 mu to 300 mu in combination with the different chamber pressures. Lyophilized cake quality was assessed by evaluating three of its properties: residual moisture, by the Karl Fischer method; the monomeric content of the protein, by RP-HPLC; and oligomeric content, by SDS-PAGE. Process uniformity was checked by determining residual moisture in cakes collected from various locations in the chamber. The experimental data show that IL-2 can be lyophilized in a pilot unit within 30 hours. The IL-2 lyophilized at various primary drying conditions retained its purity and potency throughout the stability study period (12 months).
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PMID:Lyophilization cycle development for interleukin-2. 159 83

The aerobic respiratory system of the hydrocarbonoclastic marine bacterium Pseudomonas nautica 617 ends with a single terminal oxidase. It is a heme-containing membranous protein which has been demonstrated only to reduce molecular oxygen to hydrogen peroxide [Denis, M., Arnaud S. & Malatesta, F. (1989) FEBS Lett. 247, 475-479]. The purification of this oxidase was achieved in a single step through by DEAE-Trisacryl chromatography. SDS/PAGE showed the presence of four subunits. The pI was found to be 4.45 and a Mr of 130,000 was determined by gel filtration. The amino acid composition of the purified terminal oxidase has been determined. About 52% of the residues are hydrophobic, strengthening the membranous nature of this bacterial oxidase. Room temperature optical spectra are typical of heme b with a 560-nm band for the reduced form in the alpha range. The prosthetic group is made of two hemes b, one high-spin (S = 5/2, gl = 5.9, g parallel approximately 2.0), the other low-spin (S = 1/2, gz = 2.94, gy = 2.27). No other metal centre was detected by EPR. The two hemes remained unresolved in optical spectra, even at low temperature, and throughout redox titration. They behaved potentiometrically like a one-electron, single redox couple, with Em = 87 +/- 10 mV at pH 7.2 and 293 K. The purified oxidase did not oxidize ferrocytochrome c, but displayed quinol oxidase activity both with the native quinone (2419 nmol O2.min-1.mg protein-1 and commercially available coenzyme (101.74 nmol O2.min-1.mg protein-1). Exposure of the reduced enzyme to CO induced the collapse of alpha and beta bands as occurred during reoxidation. In contrast, NaCN and NaN3 fully inhibited the oxidase activity. Results are discussed with respect to other purified quinol oxidases.
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PMID:Purification and characterization of the oxidase from the marine bacterium Pseudomonas nautica 617. 164 55

The fate of the cone-associated extracellular domain, or cone matrix sheath (CMS), was examined in two canine models of hereditary retinal degeneration. The diseases, which affect cones selectively (cd = cone degeneration), or rods and cones temporally (prcd = progressive rod-cone degeneration), were examined biochemically (SDS-PAGE/lectin blots) and cytochemically (light microscopy) using peanut agglutinin lectin (PNA) to selectively label this domain and associated structures. Most of the cones had disappeared in the adult cd retina. In the remaining cones, PNA labeled the ectopically located somata and the CMSs that were present around severely diseased ones. Loss of cones resulted in background label in the IPM and the loss of the pedicle-associated label in the OPL. SDS-PAGE of retinal extracts showed that all the major classes of the lower molecular weight PNA-binding proteins were present, but only the 40- and 60-kD bands remained prominent. Because of the selectivity of the cd mutation, this suggests considerable heterogeneity within the various size classifications of the retinal PNA-binding glycoproteins. In prcd, CMSs were normal at a time when cones were structurally normal and disease was limited to the rod outer segments. The CMSs remained intact during the degenerative phase of the disease, and only became compressed in association with the collapse and narrowing of the photoreceptor layer; CMS labeling was lost with disappearance of the cone inner segment. The lectin biochemical results were normal until 1.7 years of age; thereafter, there was a decreased prominence of all major bands. Because of spatial heterogeneity in disease severity, it was not possible to correlate the lectin biochemical and cytochemical results in prcd.
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PMID:The cone matrix sheath in the normal and diseased retina: cytochemical and biochemical studies of peanut agglutinin-binding proteins in cone and rod-cone degeneration. 185 44

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.
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PMID:Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones. 215 29

Two monoclonal antibodies, FIFI and PHIL, have been prepared using detergent-washed myogenic cells as immunogen. On Western blots of total protein extracts of muscle cells, both antibodies bind to vimentin (52 kD) and its degradation products (major band at 42 kD), but do not bind to mouse proteins or to actin (42 kD). Specificity for a determinant common to vimentin and desmin was confirmed by 2-D gel electrophoresis of muscle cell extracts and purified desmin. Western blots with FIFI reveal particularly well the extreme sensitivity of intermediate filaments (IFs) to proteolysis, which was preventable in brain tissue only by boiling in 1% SDS, although it could be reduced in both brain and muscle by less extreme methods. Western blots suggest a large increase in IF content of differentiating myoblast cell cultures at the time of cell fusion and an increase of at least 4-fold is confirmed by a quantitative immunoassay using a direct ELISA method. Immunofluorescence microscopy shows that this increase is due to the appearance of high concentrations of the intermediate filament antigen at the ends of early myotubes, preceding the appearance of cross-striations in myofibrils. Furthermore, whereas the polar filaments detected by FIFI run right to the ends of the early myotubes and only sparingly penetrate the central area, cross-striated myofibrils (as detected by the monoclonal antibody, SAM) run the length of the myotube but do not reach the ends. Colcemid and colchicine cause the vimentin filaments in fibroblasts to collapse into perinuclear rings or caps, but do not have this effect on the polar fluorescence in early myotubes. Heat shock (2 h at 45 degrees C) has a similar differential effect. The results suggest that early in muscle differentiation intermediate filament proteins accumulate rapidly at myotube ends, where they are organized differently from those in fibroblasts.
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PMID:Monoclonal antibodies to intermediate filaments in chick muscle cell cultures. 388 43

A disseminated atrophy of the proximal tubule accompanies K2HPO4-induced nephropathy in dogs. These pathologic processes cause glomerular changes that pass through different inflammatory stages and terminate in glomerular sclerosis. Experimental animals, design of the experiment and methods have been described [15]. During the 14-week study we determined the amount of urine (24 hours), protein (mg/dl), protein excretion (mg protein/24 hr) and the macro- and microprotein fraction in the urine by SDS-polyacrylamide gel electrophoresis. Clinical examinations were at 15, 66, and 85 days. Beagle dogs treated with 0.8 g K2HPO4/kg body weight developed significant glomerular selective and unselective protienuria. During the experiment the macroproteins in the urine decreased markedly, and at the last examination (day 85) glomerular proteinuria was no longer detectable by electrophoresis. Morphologically, there were only slight glomerular changes in the biopsy material taken at four weeks. Widespread lesions at 14 and 38 weeks were dilatation of Bowman's space, thickening of the basement membrane, increase in mesangial matrix, interposition of non-argentophilic mesangial matrix into the glomerular basement membrane, protein deposits in the mesangium and parietal basement membrane, formation of crescents, shrinkage of the glomeruli with collapse of glomerular tufts, and finally glomerular sclerosis. The parietal epithelial cells contained cytoplasmic areas that were free of organelles and contained microfilamentous and fine-granular material. These areas were close to the capsular basement membrane. Bundles of filaments within parietal epithelial cells had contact with the basement membrane, thus resembling hemidesmosomes. The sequelae of tubular atrophy are retention of glomerular filtrate and dilatation of Bowman's space, followed by compression and shrinkage of the glomerular tufts, and inflammatory processes within the glomerulus. The latter may be characterized as mesangio-sclerosing, mesangio-proliferative, membrano-proliferative, and extra-capillary glomerulonephritis. The decrease of urinary protein excretion towards the end of the experiment may be related to intratubular lysosomal digestion of cellular and amorphous components.
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PMID:[Potassium hydrogen phosphate induced nephropathy in the dog. II. Glomerular alterations (author's transl)]. 742 31

Mitochondria contain a structure which forms a large aqueous pore in the inner membrane after Ca2+ overload in the presence of Pi. In the present study, pore activation in liver mitochondria was monitored using the collapse of the inner membrane potential (delta psi). Ca(2+)-induced pore opening (delta psi collapse) was prevented by the immunosuppressant cyclosporin A, but cyclosporin A did not reverse pore opening (i.e. allow delta psi regeneration) unless ADP was also added. At concentrations that produced substantial pore blockade, [3H]cyclosporin partitioned more or less equally between membrane and soluble fractions, but the distribution was shifted slightly to the membranes in the presence of ADP. ADP also increased the binding of [3H]cyclosporin A to membranes washed free of soluble components. The indication that cyclosporin A inhibition of the pore is mediated by an ADP-sensitive membrane component was examined using a tritiated photoactivable derivative of cyclosporin A. ADP selectively increased covalent binding of this derivative to a membrane component. This component eluted from molecular-sizing columns as a 13-17-kDa-protein in the presence of 0.5% Chaps as detergent and migrated as a 10-kDa (approximately) protein in SDS/PAGE. These findings provide the first evidence that a protein of approximately 10 kDa may be part of the cyclosporin-A receptor of the Ca(2+)-activated pore. The possible implications of these findings are discussed.
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PMID:An ADP-sensitive cyclosporin-A-binding protein in rat liver mitochondria. 816 15

The poor axonal regeneration that follows lesions of the central nervous system (CNS) is crucially influenced by the local CNS tissue environment through which neurites have to grow. In addition to an inhibitory role of the glial scar, inhibitory substrate effects of CNS myelin and oligodendrocytes have been demonstrated. Several proteins including NI-35/250, myelin-associated glycoprotein, tenascin-R, and NG-2 have been described to have neurite outgrowth inhibitory or repulsive properties in vitro. Antibodies raised against NI-35/250 (monoclonal antibody IN-1) were shown to partially neutralize the growth inhibitory effect of CNS myelin and oligodendrocytes, and to result in long distance fiber regeneration in the lesioned adult mammalian CNS in vivo. We report here the purification of a myelin protein to apparent homogeneity from bovine spinal cord which exerts a potent neurite outgrowth inhibitory effect on PC12 cells and chick dorsal root ganglion cells, induces collapse of growth cones of chick dorsal root ganglion cells, and also inhibits the spreading of 3T3 fibroblasts. These activities could be neutralized by the monoclonal antibody IN-1. The purification procedure includes detergent solubilization, anion exchange chromatography, gel filtration, and elution from high resolution SDS-polyacrylamide gel electrophoresis. The active protein has a molecular mass of 220 kDa and an isoelectric point between 5.9 and 6.2. Its inhibitory activity is sensitive to protease treatment and resists harsh treatments like 9 M urea or short heating. Glycosylation is, if present at all, not detectable. Microsequencing resulted in six peptides and strongly suggests that this proteins is novel.
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PMID:Identification and characterization of a bovine neurite growth inhibitor (bNI-220). 966 18

The mechanism of unfolding of ferricytochrome c induced by the surfactant sodium dodecyl sulfate has been studied by heme absorption, tryptophan fluorescence, circular dichroism, resonance Raman scattering, stopped-flow and time-resolved resonance energy transfer to obtain a comprehensive view of the whole process. Unfolding occurred at an almost specific molecular ratio of SDS/cytochrome c in the concentration range (20-50 microM) studied here. However there appears to be a point at approximately 0.6 mM SDS where unfolding begins to occur for lower cytochrome c concentrations. The kinetics of unfolding revealed only a single transition with a rate constant of 33 s(-1) (at 298 K, [SDS] = 8.7 mM) and activation energy barrier of approximately 16 kJ/mol, indicating that other associated steps, if any, are too fast to be significantly populated. The free energy change (deltaG(o)) involved with the unfolding transition was estimated to be about 16.8 kJ/mol. The CD spectrum at 220 nm of SDS-unfolded cytochrome c shows only a partial decrease (25%), indicating that a significant amount of helical structure remains folded in contrast to a complete loss of helical structure in GdnHCl-denatured cytochrome c. The heme structure in SDS-unfolded cytochrome c, as deduced from heme absorption and resonance Raman spectra, shows a major population (approximately 95%) of mis-ligated histidine to the heme which acts as a kinetic trap in the folding process. The structural changes associated with cytochrome c unfolding were also monitored by time-resolved resonance energy transfer which shows a drastic increase in tryptophan fluorescence lifetime from 12 ps in the native protein to 0.63 ns in the unfolded one, associated with a movement of Trp59 by 10 A away from heme. The maximum entropy method analysis of fluorescence decay indicated the growth of various conformational substates in SDS-unfolded cytochrome c in contrast to narrowly distributed conformations in the native protein. The refolding was comprised of three kinetic steps; the first was significantly fast (approximately 8 ms) and was assigned to the dissociation of His26 that paves the protein towards correct folding pathway. The other two slower steps probably arise from chain misorganization and prolyl isomerization. The absence of a burst-phase amplitude supports the idea that the burst phase observed in the folding from completely unfolded cytochrome c corresponds to a molecular collapse that produces significant secondary structure. The partially unfolded state represents a unique intermediate state in the folding pathway.
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PMID:Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding. 968 80

Pulmonary surfactant protein A (SP-A) is an oligomeric glycoprotein that binds dipalmitoylphosphatidylcholine (DPPC). Interactions of rat SP-A and recombinant SP-As with pure and binary monolayers of DPPC and cholesterol were studied using a rhomboid surface balance at 37 degrees C. A marked inflection at equilibrium surface tension (23 mN/m) in surface tension-area isotherm of a pure DPPC film was abolished by rat SP-A. The inflection was decreased and shifted to 18 mN/m with wild-type recombinant SP-A (SP-Ahyp). Both rat SP-A and SP-Ahyp decreased surface area reduction required for pure DPPC films to reach near zero surface tension from 30 to 25%. SP-Ahyp, E195Q,R197D, mutated in carbohydrate recognition domain (CRD) known to be essential for SP-A-vesicle interactions, conveyed a detrimental effect on DPPC surface activity. SP-ADeltaG8-P80, with deletion of collagen-like domain, had little effect. Both SP-Ahyp, C6S (Ser substitution for Cys6) and SP-Ahyp,DeltaN1-A7 (N-terminal segment deletion) which appear mainly as monomers on non-reducing SDS-PAGE analysis, increased required surface area reduction for minimal surface tension. All SP-As reduced collapse surface tension of a pure cholesterol film from 27 to 23 mN/m in the presence of Ca2+. When mixed films were formed by successive spreading of DPPC/SP-A/cholesterol, rat SP-A, SP-Ahyp, or SP-ADeltaG8-P80 blocked the interaction of cholesterol with DPPC; SP-Ahyp,E195Q,R197D could not impede the interaction; SP-Ahyp,C6S or SP-Ahyp,DeltaN1-A7 only partially blocked the interaction, and cholesterol appeared to stabilize SP-Ahyp,C6S-DPPC association. These results demonstrate the importance of CRD and N-terminal dependent oligomerization in SP-A-phospholipid associations. The findings further indicate that SP-A-cholesterol interactions differ from SP-A-DPPC interactions and may be nonspecific.
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PMID:Interactions of pulmonary surfactant protein SP-A with monolayers of dipalmitoylphosphatidylcholine and cholesterol: roles of SP-A domains. 1022 61


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