Study of biochemical substrate and role of metalloproteinases in fascia transversalis from hernial processes

The aim of this study was to examine the fascia transversalis (FT) from patients with direct and indirect hernia in an attempt to identify possible differences between each type of hernia. FT samples were obtained from 36 patients presenting inguinal hernia (23 indirect hernia and 13 direct hernia) who underwent surgery. We have analysed the ultrastructure of the fascia surrounding the hernial lesions, the proline and lysine hydroxylation in the tissue, the type I-type III collagen ratio and the presence of metalloproteinases. We have not detected ultrastructural differences in the collagen fibrils from FT in direct and indirect hernias. However, the interfibrillar matrix was more abundant in direct hernias, showing abundant electron-dense particles. No differences in proline hydroxylation were observed between each type of hernia. A small decrease in lysine hydroxylation was detected in patients with direct hernia. Enzyme-linked immunosorbent assays (ELISAs) showed no statistically significant differences in the type I-type III collagen absorbance ratios. Immunohistochemistry revealed no differences in the expression of matrix metalloproteinase-1. FT from patients presenting direct hernia showed a very strong staining vs. metalloproteinase-2 when compared with that observed in indirect hernia

Characterization of hake (Merluccius merluccius L.) and trout (Salmo irideus Gibb) collagen

An analysis of the different types of collagen present in various body structures, the amino acid composition of the collagen, and the degree of collagen aggregation for hake and trout is presented. Type I collagen was found at all body sites, and its amino acid profile and degree of hydroxylation are considered. Hydroxyproline values, the ratio of hydroxyproline to the other amino acids, and the proportions of alanine, tyrosine, and methionine are also discussed. Values for collagen solubility in salt and acid indicated a lower insolubility rate for skin collagen than for muscle collagen. The proportions of the α, β, and γ components in the acid-soluble fractions of skin and muscle collagen are also examined; the proportion of γ components is lower in hake muscle collagen than in hake skin collagen, whereas the converse holds true in trout. © 1990 American Chemical Society.Peer Reviewe

Coexpression of alpha and beta subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen.

We have reported previously on the expression of recombinant human type X collagen (hrColX) in HEK 293 and HT 1080 cells by using the eukaryotic expression vector pCMVsis (in which CMV stands for cytomegalovirus). Several stably transfected clones secreted full-length triple-helical hrColX molecules in large amounts, but the secreted collagen was underhydroxylated, with a hydroxyproline-to-proline ratio of 0.25 and a melting temperature (T(m)) of 31 degrees C. By comparison, native chicken type X procollagen has a T(m) of 46 degrees C. To stabilize the triple helix of hrColX, an hrColX-expressing clone (A6/16) was co-transfected with both alpha and beta subunits of human prolyl 4-hydroxylase. Clones were selected that secreted proalpha1(X) collagen chains with an apparent molecular mass of 75 kDa and an increased hydroxyproline-to-proline ratio of close to 0.5. As a result of enhanced prolyl hydroxylation, the T(m) of the hrColX was increased to 41 degrees C as measured by CD analysis at various temperatures. The CD spectra indicated a minimum ellipticity at 198 nm and a peak at 225 nm at 20 degrees C, confirming the presence of a triple helix. The same T(m) of 41 degrees C was measured for the triple-helical core fragments of hrColX of 60-65 kDa that were retained after brief digestion with chymotrypsin/trypsin at increasing temperatures. This shows that the human cell line HEK-293 is suitable for the simultaneous expression of three genes and the stable production of substantial amounts of recombinant, fully hydroxylated type X collagen over several years

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

Use of lactic acid for extraction of fish skin gelatin

The ability of lactic acid compared to acetic acid for Dover sole (Solea vulgaris) skin swelling and the subsequent gelatin extraction was examined. The resultant gelatins were evaluated in terms of extraction yield, amino acid composition, molecular weight distribution, gel strength, viscoelastic properties, ability to refold into triple helical structures, and aggregation phenomena. Lactic acid (25 mM) proved to be an excellent substitute for acetic acid during the skin swelling process, as the gelatin preparation thus obtained presented quite similar properties to that prepared by using 50 mM acetic acid without the negative organoleptic properties of this acid. However, the application of 50 mM lactic acid gave rise to a highly hydrolysed gelatin, with lower folding ability, gel strength and viscoelastic properties than those obtained using 25 mM lactic acid or 50 mM acetic acid. © 2004 Elsevier Ltd. All rights reserved.Peer Reviewe

Epitope-specific recognition of type II collagen by rheumatoid arthritis antibodies is shared with recognition by antibodies that are arthritogenic in collagen-induced arthritis in the mouse

Objective. To analyze the fine specificity of IgG autoantibodies in sera from rheumatoid arthritis (RA) patients for type II collagen (CII) epitopes that are arthritogenic in collagen-induced arthritis (CIA), a relevant murine model of RA. Methods. For enzyme-linked immunosorbent assay (ELISA) analysis of conformation-dependent autoantibody binding, recombinant chimeric collagens that harbor the respective CII epitopes as an insertion within the frame of a constant type X collagen triple helix were constructed. In addition, synthetic peptides mimicking the native collagen structures were applied for the first time in the ELISA assessment of Immoral CII autoimmunity. Results. The pathogenicity of IgG responses to certain CII determinants in CIA was demonstrated by arthritis development in BALB/c mice upon the combined transfer of 2 mouse monoclonal antibodies specific for precisely mapped conformational CII epitopes (amino acid residues 359-369 [C1(III)] and 551-564 [J1]), whereas antibodies to another epitope (F4) were not arthritogenic. To test whether human autoimmune responses are similarly directed to these conserved CII determinants, serum IgG was analyzed. The prevalence of sera with increased IgG binding to the C1(III) epitope was significantly higher in RA compared with sera from healthy donors or from patients with other rheumatic conditions, e.g., osteoarthritis (OA), systemic lupus erythematosus (SLE), or relapsing polychondritis (RP), whereas levels of antibodies specific for the nonarthritogenic F4 epitope were associated with OA rather than RA. Conclusion. Autoimmunity to CII, although detectable in different rheumatic conditions, differs in fine specificity between distinct disease entities. In RA, in contrast to degenerative joint disease, RP, and SLE, autoantibody responses are directed to an evolutionary conserved CII structure that is also targeted by pathogenic autoimmune responses in murine models of arthritis

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 (2 h, 42 °C) and only slightly reduced by severe heat-shock (2 h, 45 °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.This work was supported by Spanish grants BMC2002-01407 and BFU2005-02671 from DGI. We are grateful to the members of the Microscopy and Flow Cytometry Center of the Complutense University of Madrid for their assistance in Confocal microscopy.Peer reviewe

Structural and physical properties of gelatin extracted from different marine species: A comparative study

Gelatin from skins of several marine species were compared on the basis of their rheological characteristics (viscoelasticity and gel strength) and chemical/structural properties (amino acid composition, molecular weight distribution and triple helix formation). Gelatins from flat-fish species (sole and megrim) presented the best gelling ability and the gels were more thermostable than those from cold-adapted fish (cod and hake). This different behaviour may be explained considering the amino acid composition, the α1/α2 collagen-chain ratio, and the molecular weight distribution. Thus, cod gelatin presented a lower alanine and imino acid content, and a decreased proline hydroxylation degree; cod and hake gelatins presented a low α1/α2 ratio (∼1); hake gelatin showed a highly significant decrease in β-components and other aggregates. The squid gelatin presented the most significant changes regarding amino acid composition and molecular weight distribution, most of these differences arising from the low solubility of the squid connective tissue. However, the squid gelatin showed viscoelastic properties intermediate between those from flat-fish and cold-adapted fish species. Circular dichroism analysis reveals that gelling involves a refolding of denatured collagen chains into the typical triple helix conformation and, conversely, unfolding upon reheating. Thermal folding and unfolding curves were similar to those of viscoelastic properties but showing a shift towards lower or higher temperatures upon cooling and heating, respectively. The folding process seem to be directly related in the stabilisation of the gels without disregarding its role in triggering the gelation process. Finally, gel strength evaluation revealed the importance of slow cold maturation. © 2001 Elsevier Science Ltd. All rights reserved.Peer Reviewe