Structural characterization and unfolding mechanism of human 4F2hc ectodomain.

4F2hc (CD98hc) is a multifunctional type II membrane glycoprotein involved in several functions as amino acid transport, cell fusion, ß1-integrin-signaling and transformation. 4F2hc ectodomain has been crystallized and its three-dimensional structure determined. We have carried out a spectroscopical/structural characterization of the recombinant ectodomain in order to obtain information on its dynamic structure in solution and on its ability to form homodimers by itself in the absence of the transmembrane helix and of the potential interactions with the plasma membrane. Analytical ultracentrifugation and crosslinking experiments showed that the ectodomain is monomeric in solution. The secondary structure determined by far-UV circular dichroism (CD) spectroscopy (around 30% α-helix and 20% ß-sheets, 12% antiparallel and 8% parallel) reveals a compact and thermally stable structure with a high melting temperature (57-59°C). Tryptophan residues are mainly buried and immobilized in the hydrophobic core of the protein as suggested by near-UV CD spectrum, the position of the Trp maximum fluorescence emission (323nm) and from the acrylamide quenching constant (2.6M(-1)). Urea unfolding equilibrium has been studied by far-UV CD and fluorescence spectroscopy to gain information on the folding/unfolding process of the ectodomain. The analyses suggest the existence of two intermediate states as reported for other TIM barrel-containing proteins rather than an independent unfolding of each domain [A, (ßα)(8) barrel; C, antiparallel ß(8) sandwich]. Folding seems to be directed by the initial formation of hydrophobic clusters within the first strands of the ß-barrel of domain A followed by additional hydrophobic interactions in domain C.

Role of anchorin CII, a 31,000-mol-wt membrane protein, in the interaction of chondrocytes with type II collagen.

We have previously reported the isolation of a hydrophobic, type-II collagen-binding glycoprotein of molecular weight 31,000 (31,000-mol-wt protein) from chick chondrocyte membranes (Mollenhauer, J., and K. von der Mark, EMBO Eur. Mol. Biol. Organ. J., 2:45-50). The function of this protein in anchoring pericellular type II collagen to the chondrocyte surface was inferred from its ability to bind native type-II collagen either when detergent solubilized or when inserted into liposomes. In the present study we have used specific antibodies to localize this protein, which we now call anchorin CII, to the surface of chondrocytes in both cartilage sections, and in cell culture. In immunofluorescence studies of isolated chondrocytes we observed a dense, punctate distribution of anchorin CII on the cell surface when chondrocytes were enclosed by a pericellular type II collagen matrix. Removal of the pericellular matrix with trypsin also removed anchorin CII. The membrane protein character of anchorin CII was indicated by the demonstration of antibody-induced patching and capping on the chondrocyte surface at 22 degrees C and 37 degrees C, respectively. In monolayer culture, the amount of anchorin CII appeared reduced on flattened chondrocytes lacking a pericellular type II collagen matrix but was prominent upon intercellular cell processes. Fab' fragments prepared from either anchorin CII antiserum or an antiserum directed against the entire chondrocyte membrane inhibited the attachment of chondrocytes to a type II collagen substrate. In each case, the inhibition of attachment was neutralized by preincubation of Fab' fragments with purified anchorin CII.

Vitreous SiO2-CaO coatings on Ti6Al4V alloys: reactivity in simulated body fluid versus osteoblast cell culture.

Vitreous coatings of the SiO(2)-CaO system have been prepared on Ti6Al4V substrates by the sol-gel method. The textural parameters (porosity and roughness) and thickness of the films obtained increase when the concentration of the precursor solutions is raised. In vitro studies of these coatings have been performed using two approaches: soaking in simulated body fluid, and by growing osteoblasts on these materials. The results of both studies show differences in terms of chemical reactivity. While in simulated body fluid the coatings were dissolved without forming a bioactive surface, when osteoblast-like cells grew on the coatings they were more stable. Furthermore, cell culture assays show biocompatible behavior of these coatings making them of potential interest for clinical applications. The effect of the textural parameters of the obtained coatings on the cell functions (attachment, spreading, proliferation and differentiation) has also been studied. The results show an increase in these cell parameters as the roughness and porosity of the coatings increase.

Biochemical and mechanical behavior of ostrich pericardium as a new biomaterial.

We have performed a comparative analysis of glutaraldehyde-preserved ostrich pericardium, as a novel biomaterial, with bovine pericardium. The biochemical characteristics (histology, water content, amino acid composition, and collagen and elastin contents), mechanical properties, and in vivo calcification in a subcutaneous rat model were examined. Ostrich pericardium is slightly thinner and shows a higher water content (70+/-2% vs. 62+/-2%) than bovine pericardium. Additionally, ostrich pericardium presents 1.6-fold lower elastin content and a lower percentage of collagen in reference to the total protein content (68+/-2% vs. 76+/-2%). However, ostrich pericardium shows better mechanical properties, with higher tensile stress at rupture (32.4+/-7.5 vs. 11.5+/-4.6) than calf pericardium. In vivo calcification studies in a rat subcutaneous model show that ostrich pericardium is significantly less calcified than bovine pericardium (23.95+/-13.30 vs. 100.10+/-37.36 mg/g tissue) after 60 days of implantation. In conclusion, glutaraldehyde-stabilized ostrich pericardium tissue shows better mechanical properties than calf tissue. However, calcium accumulation in implanted ostrich tissue is still too high to consider it a much better alternative to bovine pericardium, and anticalcification treatments should be considered.

Fatty acid synthetase complex from the insect Ceratitis capitata.

Fatty acid synthesis capacity of the insect Ceratitis capitata has been investigated in vitro from [1-14C]acetyl-CoA using homogenates at different stages of development. A maximum activity was observed after 5--6 days of larval development. But homogenates of the pharate adult insect did not show synthetic capacity of fatty acids. Fatty acid synthetase complex has been isolated from the particle-free supernatant fraction of homogenates from the 6-day C. capitata larvae. The enzyme complex was purified 182-fold with respect to the protein contained in the crude extract. The complex was homogeneous when analysed by gel filtration and by polyacrylamide-gel electrophoresis. The molecular weight was 5.2X10(5). The enzyme was dissociated into half-molecular subunits. Amino acid analysis, general properties, stability and kinetic constants (V and Km) for the substrates are reported. The fatty acid synthetase complex from the insect contains 42+/-1-SH residues and one phosphopatetheine moiety per 5.2X10(5). Activity was dependent on the presence of NADPH; FMN strongly inhibited the enzyme activity promoted by NADPH. The enzyme complex synthesized a range of fatty acid (10:0--18:0), palmitate being the predominant end product. The proportions of fatty acids synthesized varied with substrate concentrations. Fatty acids released from the complex were almost completely in the free form.

Fatty acid synthetase complex from the insect Ceratitis capitata. Structural studies.

The involvement of lipids in the structure and the activity of the fatty acid synthetase from the insect Ceratitis capitata has been previously established. Lipid-protein interactions were examined by circular dichroism. A thermal transition for both the structure and the activity of the enzyme complex takes place at about 50 degrees C; as the temperature is raised alpha-helix content decreases considerably and, concomitantly, the enzyme undergoes a marked inactivation. After 180 min at 37 degrees C, the secondary structure of the enzyme complex is 20% alpha-helix, 33% beta structure and 47% of not ordered structure against 43%, 26% and 31% as respective percentages for the native form of the complex. Lipolytic digestion of the complex was carried out with either lipase or phospholipase A2 or a mixture of both enzymes. Any of the lipolytic treatments induces a decrease of [theta]220 and the simultaneous digestion with lipase plus phospholipase during 90 min account for a limit structure with 8% of alpha-helix. The secondary structure of the complex after treatment with proteolytic enzymes, trypsin or chymotrypsin, had 15% alpha-helix, 20% beta structure and 57% of not ordered structure. The preservation of the alpha-helix content indicates that lipids protect certain of the bonds cleavable in the absence of lipids. The structural organization of the complex was studied through sequences of lipolytic and proteolytic treatments; final organization was dependent on the initial lipolytic digestion in agreement with the peptide bond shielding by the lipid component. Nitration of the complex with tetranitromethane modified almost completely all tyrosine residues of the polypeptide chains.

Midregion parathyroid hormone-related protein inhibits growth and invasion in vitro and tumorigenesis in vivo of human breast cancer cells.

Parathyroid hormone-related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post-translational processing, and PTHrP(38-94)-amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38-94)-amide in a panel of six breast cancer cell lines "in vitro" and in MDA-MB231 cells "in vivo" specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38-94)-amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38-94)-amide into MDA-MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti-breast cancer effects.

Role of the N-terminus in the structure and stability of chicken annexin V.

The role of the short N-terminal region of chicken annexin V in the maintenance of the protein structure and its influence in the conformation of the calcium binding regions was analyzed. The N-terminal domain is not essential for protein folding, wild-type and dnt-annexin V showing almost identical secondary structures. However, the partial truncation of the N-terminus significantly decreases the melting temperature of the protein and induces the partial exposure of Trp187 which is normally located in a hydrophobic pocket of the calcium binding region of domain 3 of annexin V in the Ca2+-free form.

Biocompatibility and degradability of sepiolite-collagen complex.

Sepiolite, a magnesium silicate, binds collagen resulting in a complex which has a gel-like structure when hydrated. The binding of the protein to the clay decreases its degradability by collagenase, and no degradation was observed after treatment of the complex with glutaraldehyde. Extracts of both the untreated and the glutaraldehyde-treated complex are biocompatible for fibroblast growth. Based on its properties, this material should be considered in the design of biomaterials.

Outgrowth of fibroblasts on sepiolite-collagen complex.

The studies reported here are part of a series of experiments designed to characterize connective tissue cell response to sepiolite (magnesium silicate)-collagen complexes. The process of cell migration from skin explants to these complexes, normal and glutaraldehyde-treated, is similar to the control. The relative outgrowth value for both types of complex is 1.02 and 0.90 respectively, thus indicating a normal cell outgrowth.

Gelatinases in soft tissue biomaterials. Analysis of different crosslinking agents.

Chemical modification of pericardium-based cardiac valves tends to reduce the relatively high degree of biodegradation and calcification of the implanted bioprostheses. We analysed the tissue properties of pericardium from young calves and pigs after crosslinking with different agents (glutaraldehyde. diphenylphosphorylazide (DDPA), 1-ethyl-3,3-dimethyl-aminopropyl-carbodiimide (EDAC)) and when exposed to anticalcification treatments (chloroform/methanol or ethanol) prior to glutaraldehyde (GA) crosslinking. Protein extraction after tissue homogenisation in the presence of detergents showed that crosslinking using GA or DPPA was much more effective. The amounts of protein extracted from these two groups of chemically modified pericardium were significantly lower: the other modified tissues presented only a slight reduction when compared with untreated tissue. Matrix metalloproteinases- (MMP) 2 and 9 were detected in native pericardium from calf and pig by zymography. While the MMP-9/MMP-2 activity ratio was close to 1 in pig pericardium, it was 8.5-fold higher in bovine tissue. Crosslinking with GA and with DPPA almost completely abolished gelatinase activities, even when equal amounts of solubilised protein were loaded onto the zymograms. Anticalcification treatments followed by GA crosslinking or treatment with EDAC were not as effective in reducing gelatinase activities; but, interestingly, a relative reduction of MMP-9 versus MMP-2 was detected. The presence of these gelatinase activities in pericardium may contribute to the in vivo degradability of pericardium-based cardiac valves.

Implantation of sepiolite-collagen complexes in surgically created rat calvaria defects.

The response of osseous tissue to the implantation of sepiolite-collagen complexes has been studied. Sepiolite, sepiolite-collagen complex and 0.5% glutaraldehyde-treated sepiolite-collagen complex were implanted in created circular defects in rat calvaria. The tissue reactions were analysed using light, transmission and scanning electron microscopies. The patterns of bone growth were radiographically analysed and the bone activity was indirectly quantified by using a point-count method. The reaction against the three implanted materials is characteristic of a foreign body reaction with abundant macrophages and giant cells. Implanted products have been detected in macrophages, which suggest the involvement of phagocytosis in the resorptive process. Bone grew at the implantation sites originating excrescences or sometimes a thin bridge at the defect margins. The studied materials, after implantation in contact with bone tissue, did not produce any toxic effect or necrosis, allowing bone activity.

Upregulation of annexin A1 expression by butyrate in human colon adenocarcinoma cells: role of p53, NF-Y, and p38 mitogen-activated protein kinase.

Annexin A1 is a member of a phospholipid and calcium binding family of proteins; it is involved in anti-inflammation and in the regulation of differentiation, proliferation, and apoptosis. Here, we show the existence of a functional binding site for the tumor suppressor p53 near the proximal CCAAT box and the fact that the basal expression of annexin A1 in human colon adenocarcinoma cells is driven by p53 at the transcriptional level. Posttranscriptional mechanisms may also play an important role in maintaining constitutive annexin A1 expression. In addition, a p53/NF-Y complex is detected bound to the p53 binding site on its promoter. Butyrate is a natural product of fiber degradation in the colon and a key regulator of colonic epithelium homeostasis. We show that butyrate, a class I and II histone deacetylase inhibitor, induces transcriptional activation of annexin A1 expression correlated with differentiation. The effect of butyrate is mediated through a release of NF-Y from the proximal CCAAT box and an enhancement of p53 binding. The interaction of p53 with the promoter is dependent on p38 MAPK activity either in the absence or in the presence of butyrate. Further, activation of p38 MAPK by this agent is required to increase annexin A1 promoter activity and to increase protein expression.

Acquisition of resistance to butyrate induces resistance to luminal components and other types of stress in human colon adenocarcinoma cells.

Butyrate, naturally produced by anaerobic fermentation of diet-fiber, is the major nutrient of colonocytes and also an important regulator of colonic epithelium renewal and physiology. Other luminal components, such as bile acids or bacterial products, influence these processes. The model system we used to analyze the influence of several luminal stressors is composed of a previously established cell line resistant to the apoptotic effects of butyrate and their parental butyrate-sensitive cells. Viability of butyrate-resistant cells is unaffected by mild heat-shock (2h, 42 degrees C) and only slightly reduced by severe heat-shock (2h, 45 degrees C) in contrast to their butyrate-sensitive counterparts. The higher constitutive expression of HSP70 and HSP60 could contribute to this resistance. In addition, expression of HSP70 follows a different pattern after heat-shock in both cell lines. Butyrate-resistant cells are quite unaffected by treatment with deoxycholic acid but apoptosis is induced by chenodeoxycholic acid although to a lower extent than in butyrate-sensitive cells. These resistant cells are also less sensitive to lipopolysaccharide and show differences regarding the activation of ERK following osmotic stress. Thus, the cell model herein reported is a useful tool for investigating the molecular mechanisms of resistance to apoptosis, as well as to analyze specific targets for butyrate-resistant tumors.

In vitro models for the study of the effect of butyrate on human colon adenocarcinoma cells.

The effect of butyrate has been analyzed on human colon adenocarcinoma cell lines with different properties regarding tumorigenicity, differentiation and resistance to apoptosis induced by this agent. Butyrate reduces cell proliferation, induces differentiation (according to alkaline phosphatase activity) and apoptosis, being these effects time- and concentration-dependent. The susceptibility to the cytotoxic effects of butyrate depends on the cell line considered and it is not directly related to tumorigenicity or differentiation. We show that 2mM butyrate treatment of non-tumorigenic BCS-TC2 cells for four days strongly influences the transcriptional activity, causing extensive modification in gene expression patterns (69 up-regulated and 109 down-regulated genes). Some of these genes are involved in the modulation of cell cycle progression, apoptosis and differentiation. We have analyzed the effect of butyrate in spontaneous or induced multicellular spheroids. The more stable spheroids (spontaneous or induced from butyrate-resistant cells) increase the resistance of cells to the effects of butyrate probably due to an impaired accessibility. This in vitro model could be useful to study the resistance of tumors to the effect of natural regulators (i.e. butyrate) as well as to develop and test new therapeutic approaches.

Adhesion and spreading of fibroblasts on sepiolite-collagen complexes.

In this study, adhesion, spreading, and attachment of human fibroblasts on collagen-sepiolite complexes have been studied. The behavior of the cultured cells has been compared to that of fibroblasts cultured on plastic dishes and collagen-coated dishes. This has been considered by studying the time course of the cell attachment and scanning electron microscopic observation of the events occurring during this process. The results obtained allow the conclusion that the collagen-sepiolite complexes are adhesive for cells.

Lipid requirements for the structure and function of the fatty acid synthetase complex from Ceratitis capitata.

Lipid content of purified fatty acid synthetase preparations from the Dipterous Ceratitis capitata correlated with the enzyme activity. Delipidation of the enzyme by extracting with a series of organic solvents rendered a protein without any residual activity and the treatment with phospholipase A2 for 30 min reduced the activity to 10%. Addition of lipid classes to either the native enzyme or the phospholipase-treated preparation enhanced the activity in a different manner, phosphatidylethanolamine being the most effective lipid. The role of the lipids in the lipoprotein structure of the complex was studied by circular dichroism spectra of the native enzyme and in the presence of a concentration range of urea, guanidinium chloride, sodium dodecyl sulfate, sodium cholate, and sodium chloride. 3 M urea and 1.5 M guanidinium chloride induced a conformational transition of the lipoprotein that lost its alpha-helical structure at higher concentrations of both reagents. Sodium dodecyl sulfate and sodium cholate had little effect on the alpha-helical structure, although both reagents induced the loss of enzyme activity. Cholate had essentially the same effect as phospholipids on the maintenance of the native structure but it was unable to support the enzyme activity.

Cell morphology, proliferation and collagen synthesis of human fibroblasts cultured on sepiolite-collagen complexes.

The growth and morphology as well as collagen biosynthesis of human fibroblasts obtained and cultured on sepiolite-collagen complexes have been studied. No differences on cell morphology and growth properties nor collagen synthesis were observed when compared with standard culture substrates. The type I/type III ratio of biosynthesized collagen by fibroblasts cultured on sepiolite-collagen complexes was about 5-6 with no difference when compared to control conditions. This normal behavior was also observed for the type I/type III procollagens. According to these studies the sepiolite-collagen complexes do not modify the studied features of the fibroblasts.