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

Calcium-dependent conformational rearrangements and protein stability in chicken annexin A5.

The conformational rearrangements that take place after calcium binding in chicken annexin A5 and a mutant lacking residues 3-10 were analyzed, in parallel with human annexin A5, by circular dichroism (CD), infrared spectroscopy (IR), and differential scanning calorimetry. Human and chicken annexins present a slightly different shape in the far-UV CD and IR spectra, but the main secondary-structure features are quite similar (70-80% alpha-helix). However, thermal stability of human annexin is significantly lower than its chicken counterpart (approximately 8 degrees C) and equivalent to the chicken N-terminally truncated form. The N-terminal extension contributes greatly to stabilize the overall annexin A5 structure. Infrared spectroscopy reveals the presence of two populations of alpha-helical structures, the canonical alpha-helices (approximately 1650 cm(-1)) and another, at a lower wavenumber (approximately 1634 cm(-1)), probably arising from helix-helix interactions or solvated alpha-helices. Saturation with calcium induces: alterations in the environment of the unique tryptophan residue of the recombinant proteins, as detected by near-UV CD spectroscopy; more compact tertiary structures that could account for the higher thermal stabilities (8 to 12 degrees C), this effect being higher for human annexin; and an increase in canonical alpha-helix percentage by a rearrangement of nonperiodical structure or 3(10) helices together with a variation in helix-helix interactions, as shown by amide I curve-fitting and 2D-IR

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

Histone deacetylase inhibitors upregulate MMP11 gene expression through Sp1/Smad complexes in human colon adenocarcinoma cells

AbstractMMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors

Deoxycholic and chenodeoxycholic bile acids induce apoptosis via oxidative stress in human colon adenocarcinoma cells

The continuous exposure of the colonic epithelium to high concentrations of bile acids may exert cytotoxic effects and has been related to pathogenesis of colon cancer. A better knowledge of the mechanisms by which bile acids induce toxicity is still required and may be useful for the development of new therapeutic strategies. We have studied the effect of deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA) treatments in BCS-TC2 human colon adenocarcinoma cells. Both bile acids promote cell death, being this effect higher for CDCA. Apoptosis is detected after 30 min–2 h of treatment, as observed by cell detachment, loss of membrane asymmetry, internucleosomal DNA degradation, appearance of mitochondrial transition permeability (MPT), and caspase and Bax activation. At longer treatment times, apoptosis is followed in vitro by secondary necrosis due to impaired mitochondrial activity and ATP depletion. Bile acid-induced apoptosis is a result of oxidative stress with increased ROS generation mainly by activation of plasma membrane enzymes, such as NAD(P)H oxidases and, to a lower extent, PLA2. These effects lead to a loss of mitochondrial potential and release of pro-apoptotic factors to the cytosol, which is confirmed by activation of caspase-9 and -3, but not caspase-8. This initial apoptotic steps promote cleavage of Bcl-2, allowing Bax activation and formation of additional pores in the mitochondrial membrane that amplify the apoptotic signal. Similar content being viewed by othersThis work was supported by grants BFU2005-02671 and BFU2008-04758 from the DGES (Spain).Peer reviewe

Kinetic analysis of butyrate transport in human colon adenocarcinoma cells reveals two different carrier-mediated mechanisms

International audienceButyrate has anti-tumorigenic effects on colon cancer cells, inhibits cell growth and promotes differentiation and apoptosis. These effects depend on its intracellular concentration which is regulated by its transport. We have analyzed butyrate uptake kinetics in human colon adenocarcinoma cells sensitive to the apoptotic effects of butyrate (BCS-TC2, Caco-2 and HT29), in butyrate resistant cells (BCS-TC2.BR2), and in normal colonic cells (FHC). The properties of transport were analyzed with structural analogues, specific inhibitors, and different bicarbonate and sodium concentrations. Two carrier-mediated mechanisms were detected: a low-affinity/high-capacity (K m}=109±16 mM in BCS-TC2 cells) anion exchanger and a high-affinity/low-capacity (K m}=17.9±4.0 µM in BCS-TC2 cells) proton-monocarboxylate cotransporter that was energy-dependent and activated via PKCδ. All adenocarcinoma cells analyzed express MCT1, MCT4, ancillary protein CD147, and AE2. Silencing experiments show that MCT1, whose expression increases with butyrate-treatment in butyrate-sensitive cells, plays a key role in high-affinity transport. Low-affinity uptake was mediated by a butyrate-bicarbonate antiporter along with a possible contribution of AE2 and MCT4. Butyrate treatment increased uptake in a time- and dose-dependent manner in butyrate-sensitive but not in butyrate-resistant cells. The two butyrate-uptake activities in human colon adenocarcinoma cells enable butyrate transport at different physiological conditions to maintain cell functionality. The high-affinity/low-capacity transport functions under low butyrate concentrations and may be relevant for the survival of carcinoma cells in tumor regions with low glucose and butyrate availability as well as for the normal physiology of colonocytes

Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. α-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal β-hairpin in ribotoxins and to the different length of their unstructured loops. The amino-terminal deletion mutant Δ(7–22) of α-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that α-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the β-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.This work was supported by Grant BMC2000-0551 from the Ministerio de Ciencia y Tecnología and Grants PB98–0083 and PB98–0677 from the Ministerio de Educación y Cultura