Poly-ε-caprolactone composite scaffolds for bone repair.

Synthetic biomaterials combined with cells and osteogenic factors represent a promising approach for the treatment of a number of orthopedic diseases, such as bone trauma and congenital malformations. To guarantee optimal biological properties, bone substitutes are prepared with a 3D structure and porosity grade functional to drive cell migration and proliferation, diffusion of factors, vascularization and cell waste expulsion. In this study, synthetic hydroxyapatite (HA) or rat bone extracellular matrix (BP) were examined in an effort to optimize the mechanical properties and osteogenic activity of poly-ε-caprolactone scaffolds prepared with alginate threads (PCL-AT). Using rabbit bone marrow-derived mesenchymal stem cells (rMSCs), the effects of PCL composite substrates on cell adhesion, growth and osteogenic differentiation were evaluated. Micro-CT analysis and scanning electron microscopy evidenced that porous PCL scaffolds containing HA or BP acquire a trabecular bone-like structure with interconnected pores homogenously distributed and are characterized by a pore diameter of approximately 10 µm (PCL-AT-BP) or ranging from 10 to 100 µm. Although the porosity grade of both PCL-AT-HA and PCL-AT-BP promoted optimal conditions for the cell growth of rMSCs at the early phase, the presence of BP was crucial to prolong the cell viability at the late phase. Moreover, a precocious expression of Runx2 (at 7 days) was observed in PCL-AT-BP in combination with osteogenic soluble factors suggesting that BP controls better than HA the osteogenic maturation process in bone substitutes.

In vitro evaluation of TAT-OP1 osteogenic properties and prospects for in vivo applications.

So far, osteogenic protein 1 (OP1) is biotechnologically produced and approved for the treatment of human lumbar spine fusion and long bone non-union fractures. When combined with the TAT sequence, it has been demonstrated in vitro to be easily taken up by PC12 neuronal cells and to acquire its biological activity after intracellular refolding. In this study, TAT-OP1 was shown to be a useful strategy to efficiently drive denatured OP1 into mouse MC3T3E1 pre-osteoblasts. The correct in vitro protein refolding was verified by the activation of the BMP cascade, while the osteogenic potential of OP1 was demonstrated by increased expression of alkaline phosphatase, osteonectin and osteocalcin.

Photoactivation of corticosteroids in UVB-exposed skin.

The photodegradation of flumethasone (FM) and fluocinolone acetonide (FC) was studied in solution and in the pig skin. Both glucocorticosteroids applied to the pig skin were unstable under UVB light. The photoproducts formed in the skin were the lumi-, photolumi- and andro-derivatives for FM, the same found in vitro. Instead, FC hydroperoxide formed in solution was not found in the skin: the reactivity and oxidative ability of this photoproduct towards biological substrates (lipids, proteins) seems the reason of the lack of its detection in the ex vivo model. In fact, it demonstrated to quickly oxidize amino acids and peptides, and to react with BSA both in the dark and under irradiation. Moreover, the presence in the irradiated pig skin of the FC andro-derivative, which usually forms in H-donating environment, seems consistent with the mechanism of Norrish I fragmentation followed by H-abstraction, likely from the surrounding biological substrates. These findings indicate that photoreactivity of these compounds may take place in the skin of patients exposing themselves to sunlight and is a warning about possible skin damage as a result of that. Furthermore, photolability of these drugs in the skin might cause loss of their therapeutic activity.

Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells.

Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.

In vitro biological activity of bovine milk ribonuclease-4.

Several members of the ribonuclease superfamily possess a variety of interesting biological properties, including ribonucleolytic, angiogenic, antiproliferative, cytotoxic, embryotoxic, aspermatogenic and antitumoral activity. In this study, we report the purification from bovine milk of a protein with structural and enzymatic properties very similar to those of ribonuclease-4 (RNase-4), which is normally present in the liver and lungs, and examined its functional properties, biological activity and cytotoxic effects. RNase-4, at physiological concentrations, had a positive effect on the vitality and proliferation of human umbilical vein endothelial cells. Moreover, it induced an increase in cellular migration and the formation of in vitro capillary-like structures. We also evaluated the effect of RNase-4 in vitro on human breast, colorectal and cervical carcinoma cell lines. The protein was revealed to have a cytotoxic effect similar to that of RNase-A. We suggest that the positive effects of RNase-4 on normal cells were due to its particularly close interaction with RNase inhibitor, while good conformational stability and resistance to proteolytic degradation potentially favour ribonuclease cytotoxicity.

SP-A, SP-B, and SP-C in surfactant subtypes around birth: reexamination of alveolar life cycle of surfactant.

Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.

Mass spectrometric approaches in structural identification of the reaction products arising from the interaction between glucose and lysine.

The products arising from the reaction of alpha-protected lysine with glucose have been studied by different techniques, viz. high-performance liquid chromatography (HPLC) with UV detection, fast atom bombardment (FAB) mass spectrometry (MS), and HPLC/MS. Most of the analytical data were obtained by the last approach and allowed identification of many molecular species for a thorough knowledge of possible reaction pathways or structural data already available in the literature.

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin.

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule.

The life cycle of a low-molecular-weight protein of surfactant (SP-C) in 3-day-old rabbits.

To clarify the metabolic cycle of a low-molecular-weight protein of surfactant (SP-C), we obtained alveolar surfactant from 3 day old rabbits killed 24 h after the tracheal administration of 32P or L-[35S]methionine (donors). Aliquots of this naturally labelled surfactant were administered into trachea to 3-day-old rabbits (recipients) which were killed after 1 min or 3, 8 or 24 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine in fractions of lung lavage fluid and in lung homogenate. We found that alveolar SP-C is turned over faster than saturated phosphatidylcholine, that alveolar macrophages do participate in the removal of SP-C and that SP-C does not enter the fraction of alveolar surfactant that remains unsedimented after ultracentrifugation. Considering the whole lung, SP-C and saturated phosphatidylcholine are turned over at a comparable speed.

Autolysis of thermolysin. Isolation and characterization of a folded three-fragment complex.

Incubation of the neutral metalloendopeptidase thermolysin at pH 7.2 in the presence of EDTA and/or low concentrations of calcium ions produces fast enzyme inactivation as a result of autolysis. The 'nicked' protein is a folded species composed of three tightly associated protein fragments. Dissociation of this complex can be achieved under denaturing conditions, such as gel filtration on a column equilibrated with 5 M guanidine hydrochloride or reverse-phase high-performance liquid chromatography (HPLC) at acidic pH. The positions of the peptide bond cleavages were defined by isolation of the individual fragments by HPLC and their characterization by amino acid analysis after acid hydrolysis, end-group determination and partial amino acid sequencing. The results of these analyses indicated that the nicked protein is composed of fragments 1-196, 197-204 and 205-316 and thus that the corresponding sites of limited proteolysis occur at the polypeptide chain loop involved in the binding of Ca(4) in native thermolysin [Matthews, B. W., Weaver, L. H. and Kester, W. R. (1974) J. Biol. Chem. 249, 8030-8044]. The overall conformational properties of nicked thermolysin are quite similar to those of the intact protein, as judged by spectroscopic measurements and by the fact that rabbit antibodies against native thermolysin recognize and precipitate the nicked protein in immunodiffusion assays. The nicked protein was much less stable to heat and unfolding agents than intact thermolysin. These results contribute to a better knowledge of the molecular mechanism of stabilization of native thermolysin by the four bound calcium ions and demonstrate that the function of Ca(4) is to stabilize the loop 190-205 on the surface of the molecule against autolysis.

Correlation between sites of limited proteolysis and segmental mobility in thermolysin.

Limited proteolysis or autolysis of thermolysin under different experimental conditions leads to fission of a small number of peptide bonds located in exposed surface segments of the polypeptide chain characterized by highest mobility, as given by the temperature factors (B values) determined crystallographically [Holmes, M.A., & Matthews, B.W. (1982) J. Mol. Biol. 160, 623-639]. Considering also similar findings observed previously with other protein systems, it is proposed that this correlation between segmental mobility and sites of limited proteolysis in globular proteins is quite general. Thus, flexibility of the polypeptide chain of a globular protein at the site of proteolytic attack promotes optimal binding and proper interaction with the active site of the protease. These findings emphasize that apparent thermal motion seen in protein crystals is relevant to motion in solution and appear to be of general significance in protein-protein recognition processes.

Further characterization and amino acid sequence of m-type thioredoxins from spinach chloroplasts.

The complete primary structure of m-type thioredoxin from spinach chloroplasts has been sequenced by conventional sequencing including fragmentation, Edman degradation and carboxypeptidase digestion. As already reported [Tsugita, A., Maeda, K. & Schürmann, P. (1983) Biochem. Biophys. Res. Commun. 115, 1-7] these thioredoxins contain the same active-site sequence as thioredoxins from other sources. Based on the amino acid sequence thioredoxin mc contains 103 residues, has a relative molecular mass of 11425 and a molar absorption coefficient at 280 nm of 19 300 M-1 cm-1. The spinach thioredoxin mc has an overall homology of 44% with the thioredoxin from Escherichia coli mainly due to differences in the N-terminal and C-terminal regions.

Properties of Sepharose-bound beta-lactamase from Enterobacter cloacae.

beta-Lactamase from Enterobacter cloacae P99 was immobilized onto Sepharose by the cyanogen bromide activation method and the properties of the Sepharose-bound enzyme were compared with those of soluble and cell-bound enzyme. The immobilized beta-lactamase showed enhanced stability to storage at 4 degrees C (approximately 1 year) in respect to the free enzyme in solution (few days). The optimum pH for activity is similar for both Sepharose- and cell-bound beta-lactamase and extends over a broader pH range (pH 6-9) than the soluble enzyme (pH 8-9). Immobilization leads also to significant enhancement of thermal stability. Effective enzyme inhibition by flucloxacillin occurs with both soluble and Sepharose-bound beta-lactamase, whereas the cell-bound enzyme is much less (10(-5) times) inhibited. These results indicate that immobilized beta-lactamase could be usefully employed as a tool for investigating the properties of newly designed beta-lactamase inhibitors.

Limited proteolysis of thermolysin by subtilisin: isolation and characterization of a partially active enzyme derivative.

Incubation of the neutral metalloendopeptidase thermolysin at pH 9-10 in the presence of 10 mM CaCl2 for 2 days at room temperature with subtilisin at a 50:1 molar ratio leads to a derivative possessing lower (approximately 3%) but intrinsic catalytic activity. This derivative, called thermolysin S, was isolated by gel filtration in approximately 80% yield and then separated from some residual intact thermolysin by an affinity chromatographic step on Sepharose-Gly-D-Phe. It was found that thermolysin S results from a tight association of two polypeptide fragments of apparent Mr of 24000 and 10000. Dissociation of the complex was achieved under strong denaturing conditions, such as gel filtration on a column equilibrated and eluted with 5 M guanidine hydrochloride. The positions of the clip sites were defined by amino acid analysis, end-group determination, and amino acid sequencing of the isolated fragments and shown to lie between Thr-4 and Ser-5, between Thr-224 and Gln-225, and also between Gln-225 and Asp-226. Thermolysin S, which is therefore a stable complex of fragments 5-224(225) and 225(226)-316, shows a shift in optimum pH of about 1 unit toward the acid range with respect to intact thermolysin and a Km essentially unchanged, with furylacryloyl-Gly-Leu-NH2 as substrate. Inhibitors of thermolysin such as ethoxyformic anhydride and Zn2+ ions inactivate also the nicked enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and properties of an all-D model ribonuclease S-peptide.

In order to examine the effect of a defined enantiomeric sequence on protein structure, the all-D model ribonuclease S-peptide, H-Ala-Glu-Ala4-Lys-Phe-Ala-Arg-Ala-His-Met-Ala2-OH, has been synthesized by the solid phase method. The all-L peptide has been synthesized previously and shown to possess 36% of ribonuclease S activity when added to ribonuclease S-protein (Komoriya, A. & Chaiken, I.M. (1982) J. Biol. Chem 257, 2599-2604). The synthetic D-peptide was purified by gel filtration and semipreparative reverse phase HPLC. Amino acid composition of the synthetic peptide was in agreement with theory and gas chromatographic analysis showed that no significant racemization had occurred during synthesis. Circular dichroism (CD) studies of the D-peptide showed a peak of positive ellipticity in the 220-230 nm region, whereas a negative ellipticity peak for the L-peptide was observed. The effects of temperature and trifluoroethanol on the far-ultraviolet CD spectra of D- and L-peptides were similar but of opposite sign, confirming the expectation that the D-peptide has the propensity to form an alpha-helical structure which is enantiomeric with respect to that formed by the L-peptide. In the presence of S-protein, the L-peptide showed hydrolytic activity against the substrate cytidine-2':3'-monophosphate, whereas the D-peptide was inactive. Addition of the D-peptide to mixtures of L-peptide and S-protein did not lead to inhibition of enzymatic activity. These results indicate lack of binding of D-peptide to S-protein to produce either an active or inactive species.

Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure.

The COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin.

Evidence for coiled-coil alpha-helical regions in the long arm of laminin.

Three new laminin fragments, E8, E9 and 25K with mol. wt. 50 000-280 000, were prepared from a limited elastase digest of laminin and from tissue extracts. They were similar with respect to their rod-like structure, a high alpha-helix content, the assembly from two chain segments and immunological cross-reactivity. Two of the fragments (E8 and E9) possess in addition globular domains which lack alpha-helices. Chemical, immunological and physical data together with sequence analysis strongly indicate that the alpha-helical segments are assembled in coiled-coil structures which are located in the rod of the long arm of laminin. These data give new insights into the overall structure of the protein.

Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin.

The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of large peptides derived by cyanogen bromide cleavage of thermolysin using fast protein liquid chromatography (FPLC).

The recently introduced fast protein liquid chromatography (FPLC) system of Pharmacia (Uppsala, Sweden) was employed to isolate rather large peptides derived from thermolysin by selective chemical fragmentation at methionine in positions 120 and 205 of the polypeptide chain of 316 amino acid residues. Thermolysin was cleaved under conditions of limited fragmentation in order to produce, besides fragments 1-120, 121-205 and 206-316, the overlapping fragments 1-205 and 121-316. These polypeptides were separated employing prepacked Mono Q or Mono S columns (quaternary ammonium and sulfonic acid support, respectively). The columns were equilibrated with acetate-7 M urea buffer, pH 5.0 or 6.0, and eluted with a gradient of sodium chloride or acetate. Separations were achieved in 10-20 min and were carried out also at a semi-preparative level (1-3 mg per run). All five protein fragments were isolated in homogeneous form, as judged by amino acid analysis and electrophoresis. Considering that protein fragmentation with cyanogen bromide is the most commonly used procedure to achieve selective chemical fragmentation of a polypeptide chain, these results indicate that FPLC with ionic exchangers can be usefully employed to isolate rather large protein fragments especially suitable for automatic sequence analysis with the sequenator.

Spectroscopic study on the structure and stability of beef liver arginase.

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.