Isolation and characterization of thermostable collagen from the marine eel-fish (Evenchelys macrura)

Aim and methodsCollagen is the most abundant protein found in animal body, which is widely used for biomedical and pharmaceutical applications. In the present study, acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin wastes of marine eel fish (Evenchelys macrura) were isolated and characterized.ResultsASC and PSC extracted from eel fish skin showed the yields of 80 and 7.10 percent (based on dry weight), respectively. ASC and PSC comprising different α-chains (α1, α2 and α3) were characterized as type I and exhibited high solubility in acidic pH (1–4) and were soluble in the presence of NaCl at concentration up to 3.0 and 4.0 percent (w/v) for ASC and PSC, respectively. Amino acids analysis of both ASC and PSC contained imino acid of 190 and 200 residues per 1000 residues, respectively. The present results of ASC and PSC from eel fish skin exhibited higher thermal stability of 39 °C and 35 °C, respectively. Similar, Fourier transform infrared (FTIR) spectra of ASC and PSC were observed and suggesting that pepsin hydrolysis did not affect the secondary structure of collagen, especially triple-helical structure.ConclusionThese results suggest that the marine eel fish skin collagen close to the T_d (denaturation temperature) of mammalian collagen which could be used in the biomedical materials, food and pharmaceutical industries as an alternative source.     

Isolation and characterization of collagen from the cartilage of Amur sturgeon (Acipenser schrenckii)

The collagen in Amur sturgeon cartilage was isolated using sodium chloride (salt-solubilized collagen, SSC, 2.18%), followed by acetic acid (acid-solubilized collagen, ASC, 27.04%) and then pepsin (pepsin-solubilized collagen, PSC, 55.92%). These collagens appeared to be dense sheet-like film linked by random-coiled filaments under SEM. The denaturation and melting temperatures of PSC (35.71 and 123.90 °C) were significantly higher than SSC (32.64 and 114.51 °C) and ASC (32.98 and 120.72 °C) assessed by circular dichroism and differential scanning calorimetry, which could be attributed to its high imino acid content (22.57%) and degree of hydroxylation (47.29%). Electrophoresis pattern showed that the SSC and ASC were type I collagen, while PSC was predominantly type II collagen along with other minor types. Infrared spectra confirmed their triple helical structure, and indicated more hydrogen bonding in ASC and more intermolecular crosslinks in PSC. These results provide some basis for their large-scale production and further application as alternatives to mammalian collagen.     

Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar

Extraction of bromelain from pineapple peel (Nang Lae cultv.) using aqueous two phase system (ATPS) was optimized. Some biochemical properties including collagen hydrolysis were also investigated. Bromelain predominantly partitioned to the polyethylene glycol (PEG)-rich phase. The highest enzyme activity recovery (113.54%) and purification fold (2.23) were presented in the top phase of 15% PEG2000–14% MgSO₄. Protein pattern and activity staining showed the molecular weight (MW) of bromelain to be about 29 kDa. The extracted bromelain showed the highest relative activity at pH 7.0 and 55 °C. Its activity was decreased continuously by increasing NaCl concentration (up to 1.5% (w/v)). The bromelain extract was applied to hydrolyze the skin collagen of beef and giant catfish (0–0.3 units). The β, α₁, α₂ of giant catfish skin collagen extensively degraded into small peptides when treated with 0.02 units of the bromelain extract. Bovine collagen was hydrolyzed using higher bromelain up to 0.18 units. This study showed the ATPS can be employed to partially purify bromelain from Nang Lae pineapple peel and the enzyme effectively hydrolyzed the collagens.     

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. ¹H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.     

Separation of β22 dimer from bovine bone collagen

The precise role of the α₂-chain in collagen type I is of considerable scientific interest. Our recent studies demonstrated that the most noticeable difference between type I collagens, which were obtained from bovine hard tissues (bone, dentine) and soft tissues (tendon, skin), was presented in the position of β chain dimers using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The additional band observed both in the bone and dentine collagen was putatively identified as β₂₂ dimer (made of by an intermolecular cross-linking between two α₂-chains). Further investigations carried out on bovine bone and skin collagen, corresponding to hard tissue and soft tissue collagen respectively, confirmed this hypothesis. Successful separation of individual β₂₂ dimer from bone collagen was achieved. The procedure involves molecular-sieve chromatography on a Sephacryl S-400 column followed by differential acetone precipitation. Identification was done by the widely used methods, such as SDS-PAGE and cyanogen bromide (CNBr)-cleaved peptide analysis. It was proposed that the dimer and consequently α₂-chains may play important roles in the morphological and biological differences between hard and soft tissues.     

A novel fibrinolytic metalloproteinase,barnettlysin-I from Bothrops barnetti (barnett´s pitviper) snake venom with anti-platelet properties

BackgroundViperid snake venoms contain active components that interfere with hemostasis. We report a new P-I class snake venom metalloproteinase (SVMP), barnettlysin-I (Bar-I), isolated from the venom of Bothrops barnetti and evaluated its fibrinolytic and antithrombotic potential.MethodsBar-I was purified using a combination of molecular exclusion and cation-exchange chromatographies. We describe some biochemical features of Bar-I associated with its effects on hemostasis and platelet function.ResultsBar-I is a 23.386 kDa single-chain polypeptide with pI of 6.7. Its sequence (202 residues) shows high homology to other members of the SVMPs. The enzymatic activity on dimethylcasein (DMC) is inhibited by metalloproteinase inhibitors e.g. EDTA, and by α2-macroglobulin. Bar-I degrades fibrin and fibrinogen dose- and time-dependently by cleaving their α-chains. Furthermore, it hydrolyses plasma fibronectin but not laminin nor collagen type I. In vitro Bar-I dissolves fibrin clots made either from purified fibrinogen or from whole blood. In contrast to many other P-I SVMPs, Bar-I is devoid of hemorrhagic activity. Also, Bar-I dose- and time-dependently inhibits aggregation of washed human platelets induced by vWF plus ristocetin and collagen (IC₅₀ = 1.3 and 3.2 μM, respectively), presumably Bar-I cleaves both vWF and GPIb. Thus, it effectively inhibits vWF-induced platelet aggregation. Moreover, this proteinase cleaves the collagen-binding α2-A domain (160 kDa) of α2β1-integrin. This explains why it additionally inhibits collagen-induced platelet activation.ConclusionA non-hemorrhagic but fibrinolytic metalloproteinase dissolves fibrin clots in vitro and impairs platelet function.General significanceThis study provides new opportunities for drug development of a fibrinolytic agent with antithrombotic effect.     

Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts

Ascorbic acid (AA) is essential for collagen biosynthesis as a cofactor for prolyl and lysyl hydroxylase and as a stimulus for collagen gene expression. Many studies have evaluated the relationship between AA and collagen expression in short- and long-term effects on cells after a single administration of AA into the culture medium. However, no such study has monitored in detail the stability of AA in medium or the alterations of intracellular AA levels during a protracted interval. Therefore, we examined here intracellular AA levels and stability throughout its exposure to human skin fibroblasts in vitro. Moreover, we determined the effects on type 1 and type 4 collagen and sodium-dependent vitamin C transporter (SVCT) gene expression when medium containing 100 μM AA was replaced every 24 h for 5 days to avoid depletion of AA. Throughout this long-term culture, intracellular AA levels remained constant; the expression of type 1 and type 4 collagens and SVCT2 mRNA was enhanced, and type 1 procollagen synthesis increased. Thus, these results indicate that human skin fibroblasts exposed to AA over time had rising levels of type 1/type 4 collagens and SVCT2 mRNA expression and type 1 procollagen synthesis.     

Lobeline esters as novel ligands for neuronal nicotinic acetylcholine receptors and neurotransmitter transporters

Vesicular monoamine transporter-2 (VMAT2) is a viable target for development of pharmacotherapies for psychostimulant abuse. Lobeline (1) is a potent antagonist at α4β21 nicotinic acetylcholine receptors, has moderate affinity (K_i = 5.46 μM) for VMAT2, and is being investigated currently as a clinical candidate for treatment of psychostimulant abuse. A series of carboxylic acid and sulfonic acid ester analogs 2–20 of lobeline were synthesized and evaluated for interaction with α4β21 and α71 neuronal nicotinic acetylcholine receptors (nAChRs), the dopamine transporter (DAT), serotonin transporter (SERT) and VMAT2. Both carboxylic acid and sulfonic acid esters had low affinity at α71 nAChRs. Similar to lobeline (K_i = 4 nM), sulfonic acid esters had high affinity at α4β21 (K_i = 5–17 nM). Aromatic carboxylic acid ester analogs of lobeline (2–4) were 100–1000-fold less potent than lobeline at α4β21 nAChRs, whereas aliphatic carboxylic acid ester analogs were 10–100-fold less potent than lobeline at α4β21. Two representative lobeline esters, the 10-O-benzoate (2) and the 10-O-benzenesulfonate (10) were evaluated in the ³⁶Rb⁺ efflux assay using rat thalamic synaptosomes, and were shown to be antagonists with IC₅₀ values of 0.85 μM and 1.60 μM, respectively. Both carboxylic and sulfonic acid esters exhibited a range of potencies (equipotent to 13–45-fold greater potency compared to lobeline) for inhibiting DAT and SERT, respectively, and like lobeline, had moderate affinity (K_i = 1.98–10.8 μM) for VMAT2. One of the more interesting analogs, p-methoxybenzoic acid ester 4, had low affinity at α4β21 nAChRs (K_i = 19.3 μM) and was equipotent with lobeline, at VMAT2 (K_i = 2.98 μM), exhibiting a 6.5-fold selectivity for VMAT2 over α4β2 nAChRs. Thus, esterification of the lobeline molecule may be a useful structural modification for the development of lobeline analogs with improved selectivity at VMAT2.     

Characteristics of Korean-Saanen goat milk caseins and somatic cell counts in comparison with Holstein cow milk counterparts

Characteristics of α- and β-casein fractions in the milk of Korean-Saanen goats were compared with those of Holstein cow milk using capillary electrophoresis (CE) analysis. The αs₁-CN content of the Saanen goat milk samples varied from 2.4% to 9.3% of total proteins. Total αs-CN content of the goat milk varied from 10.1% to 17.0%. Total β-CN content containing β₁-CN and the β₂-CN varied from 49.6% to 61.0% of total proteins. Average αs-CN to β-CN ratio of the Saanen goat milk from different farms was 0.24 ± 0.04, ranging from 0.17 to 0.33. The αs-CN (αs₁-CN + αs₀-CN) to β-CN (β_A1-CN + β_A2-CN) ratio of Holstein cow milk was 0.81, which was much higher than that of Korean-Saanen goat milk. The goat milk samples having more than 1.5 million cells/ml somatic cell counts (SCC) contained higher αs-CNs (P < 0.01) and lower β-CNs (P < 0.05) contents than milks with <1.5 million SCC. This resulted in a higher αs-CN to β-CN ratio (P < 0.01) in the milk with >1.5 million SCC.     

Pentacyclic triterpenes as α-glucosidase and α-amylase inhibitors: Structure-activity relationships and the synergism with acarbose

In this paper, the inhibition of α-amylase and α-glucosidase by nine pentacyclic triterpenes was determined. For α-amylase inhibitory activity, the IC₅₀ values of ursolic acid, corosolic acid, and oleanolic acid were 22.6 ± 2.4 μM, 31.2 ± 3.4 μM, and 94.1 ± 6.7 μM, respectively. For α-glucosidase inhibition, the IC₅₀ values of ursolic acid, corosolic acid, betulinic acid, and oleanolic acid were 12.1 ± 1.0 μM, 17.2 ± 0.9 μM, 14.9 ± 1.9 μM, and 35.6 ± 2.6 μM, respectively. The combination of corosolic acid and oleanolic acid with acarbose showed synergistic inhibition against α-amylase. The combination of the tested triterpenes with acarbose mainly exhibited additive inhibition against α-glucosidase. Kinetic studies revealed that corosolic acid and oleanolic acid showed non-competitive inhibition and acarbose showed mixed-type inhibition against α-amylase. The results provide valuable implications for the triterpenes (ursolic acid, corosolic acid, and oleanolic acid) alone or in combination with acarbose as a therapeutic agent for the treatment of diabetes mellitus.     

Tissue viscoelasticity is related to tissue composition but may not fully predict the apparent-level viscoelasticity in human trabecular bone – An experimental and finite element study

Trabecular bone is viscoelastic under dynamic loading. However, it is unclear how tissue viscoelasticity controls viscoelasticity at the apparent-level. In this study, viscoelasticity of cylindrical human trabecular bone samples (n = 11, male, age 18–78 years) from 11 proximal femurs were characterized using dynamic and stress-relaxation testing at the apparent-level and with creep nanoindentation at the tissue-level. In addition, bone tissue elasticity was determined using scanning acoustic microscope (SAM). Tissue composition and collagen crosslinks were assessed using Raman micro-spectroscopy and high performance liquid chromatography (HPLC), respectively. Values of material parameters were obtained from finite element (FE) models by optimizing tissue-level creep and apparent-level stress-relaxation to experimental nanoindentation and unconfined compression testing values, respectively, utilizing the second order Prony series to depict viscoelasticity. FE simulations showed that tissue-level equilibrium elastic modulus (E_eq) increased with increasing crystallinity (r = 0.730, p = .011) while at the apparent-level it increased with increasing hydroxylysyl pyridinoline content (r = 0.718, p = .019). In addition, the normalized shear modulus g₁ (r = −0.780, p = .005) decreased with increasing collagen ratio (amide III/CH₂) at the tissue-level, but increased (r = 0.696, p = .025) with increasing collagen ratio at the apparent-level. No significant relations were found between the measured or simulated viscoelastic parameters at the tissue- and apparent-levels nor were the parameters related to tissue elasticity determined with SAM. However, only E_eq, g₂ and relaxation time τ₁ from simulated viscoelastic values were statistically different between tissue- and apparent-levels (p < .01). These findings indicate that bone tissue viscoelasticity is affected by tissue composition but may not fully predict the macroscale viscoelasticity in human trabecular bone.     

Two stages of treatments for upgrading bleached softwood paper grade pulp to dissolving pulp for viscose production

To convert bleached softwood paper grade pulp into dissolving pulp for viscose application, two stages of treatments consisting of enzymatic treatment and alkaline peroxide treatment were investigated. It was found that high reactivity (about 80%) of pulp could be achieved by endoglucanases (EG)-rich industrial cellulase treatment, and the α-cellulose content as well as the viscosity of enzymatically treated pulp can be further adjusted by the alkaline peroxide treatment with certain dosages of NaOH and H₂O₂ to finally meet the quality requirements of dissolving pulp. The resulting pulp with 68.7% of reactivity, 92.1% of α-cellulose content, and 506.9 mL/g of pulp viscosity could be obtained after the two stages of treatments. The appropriate dosage of EG-rich cellulase was 300 IU/g bone dry pulp in the stage of enzymatic treatment, while the suitable dosages of NaOH and H₂O₂ were 9 wt% and 1 wt%, respectively, in the stage of alkaline peroxide treatment.     

Immunological effects of collagen and collagen peptide from blue shark cartilage on 6T-CEM cells

The physicochemical and in vitro mechanism of immunologic tolerance of pepsin-soluble collagen and its peptide, CII-P, from blue shark cartilage were studied. Protein patterns showed three identical (α₁)₃ chains, suggesting that it was a type-II collagen (CII). CII-P had high antioxidant activity and low carbohydrate content. Collagens had better biocompatibility with decreased the viability of 6T-CEM cell compared to control cells (without collagen). Immunological indices such as FAS/APO-1, cytokine, and caspase levels were higher in CII-treated 6T-CEM cells. Collagen bound to 6T-CEM cell receptors in a dose-dependent manner, and an optimum effect was observed with 10 μg/mL collagen. The high carbohydrate content of CII could activate the FAS receptor, which led to increased apoptotic gene expression in 6T-CEM cells. Breakdown of 6T-CEM cell nuclei through the induction of apoptosis by CII was confirmed by fluorescence microscopy. Collagen molecular weight and glycosylation patterns were crucial factors for immunologic tolerance and 6T-CEM cellular apoptosis.     

Synthesis and biological evaluation of piperlongumine derivatives as potent anti-inflammatory agents

Piperlongumine (PL) and its derivatives were synthesized by the direct reaction between acid chloride of 3,4,5-trimethoxycinnamic acid and various amides/lactams. Later their anti-inflammatory effects were evaluated in lipopolysaccharide (LPS)-induced RAW-264.7 macrophages. Of the piperlogs prepared in this study, the maximum (91%) inhibitory activity was observed with PL (IC₅₀ = 3 μM) but showed cytotoxicity whereas compound 3 (IC₅₀ = 6 μM) which possess α,β-unsaturated γ-butyrolactam moiety offered good level (65%) of activity with no cytotoxicity. This study revealed that amide/lactam moiety connected to cinnamoyl group with minimum 3 carbon chain length and α,β-unsaturation is fruitful to show potent anti-inflammatory activity.     

Interactive effects of α-NAA and UV-B radiation on the endogenous hormone contents and growth of Trichosanthes kirilowii Maxim seedlings

The impact of UV-B radiation on endogenous hormones in plants has recently drawn attention from researchers. The mechanism for reduced stem elongation by UV-B might be due to changes in the phytohormone levels, especially IAA, which plays a role in stem elongation. In this study, effects of UV-B radiation on Trichosanthes kirilowii Maxim (T. kirilowii) seedlings in greenhouse-grown plants were investigated. The results indicated that: (1) In comparison to controls, exposure to 0.029 Jm^?2 s^?1. UV-B radiation led to accumulation of endogenous abscisic acid (ABA) and zeatinriboside (ZR) in the plant contents, and decreased contents of endogenous indole-3-acetic acid (IAA) and gibberellic acid (GA_1/3). Exposure to UV-B radiation reduced the height and leaf area of plants. As a result, total biomass (plant dry weight) was lower. (2) In comparison to controls, addition of 2 mg l^?1 α-naphthaleneacetic acid (α-NAA) slightly increased the contents of IAA, GA_1/3 and ZR, and decreased the content of ABA in leaves. This addition of α-NAA significantly increased plant height and leaf area, but only slightly increased total biomass. (3) Addition of α-NAA to UV-B-exposed plants: increased the content of endogenous IAA, GA_1/3 and ZR; decreased accumulation of endogenous ABA; and increased plant height and leaf area in comparison to plants that only were exposed to UV-B. Moreover, total biomass increased slightly. This suggests that addition of α-NAA may compensate to a certain extent for the lack of IAA resulting from UV-B radiation; it also increases the content of GA_1/3 and ZR, decreases the accumulation of ABA, and promotes the growth of plants.     

Enzymatic transformation of 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) by immobilized α-cyclodextrin glucanotransferase from recombinant Escherichia coli

This work aims to produce 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin with immobilized α-cyclodextrin glucanotransferase (α-CGTase) from recombinant Escherichia coli. Molecular sieve (SBA-15) was used as an adsorbent, and sodium alginate was used as a carrier, and glutaraldehyde (GA) was used as a cross-linker. The effects of several key variables on α-CGTase immobilization were examined, and optimal immobilization conditions were determined as the following: glutaraldehyde (GA, cross-linker) 0.01% (v/v), SBA-15 (adsorbent) 2 g/L, CaCl₂ 3 g/L, sodium alginate 20 g/L, adsorption time 3 h, and immobilization time 1 h. In comparison with free α-CGTase, immobilized α-CGTase had a similar optimal pH (5.5) and a higher optimal temperature (45 °C). The continuous production of AA-2G from ascorbic acid and β-cyclodextrin in the presence of immobilized α-CGTase was carried out, and the highest AA-2G production reached 21 g/L, which was 2-fold of that with free α-CGTase. The immobilization procedure developed here was efficient for α-CGTase immobilization, which was proved to be a prospective approach for the enzymatic production of AA-2G.     

Seasonal variations in fatty acid composition of pasture forage plants and CLA content in ewe milk fat

The relations between fatty acids (FAs) composition of pasture forage plants and the content of conjugated linoleic acid (CLA) as a total content of cis-9, trans-11 + trans-7, cis-9 + trans-8, cis-10 CLA isomers in ewes’ milk fat during natural pasture season (April–September) were investigated. The extracts of ewes’ milk fat samples as well as the pasture samples were analyzed for fatty acid composition by capillary gas chromatography with flame ionization and mass spectrometric detection. α-Linolenic, linoleic, and palmitic acids were predominant in pasture plants, and their contents varied during pasture season. The most abundant and most varied fatty acid compound in pasture plants was α-linolenic acid. Its content significantly decreased from 62% to 39% (of total FA) (P < 0.001) from May to August, and subsequently it slightly (57%) increased from August to September (P < 0.05), compared with the beginning of pasture season. Similarly, the content of CLA in ewes’ milk fat decreased from 2.4% in May to 1.3% in August (P < 0.001), and subsequently it rose to 2.6% in September (P < 0.001). The α-linolenic/linoleic acid ratio in the pasture sample decreased from 4.36 in May to 1.97 in August (P < 0.001), and subsequently it increased to 3.14 in September (P < 0.001); thus, it reached the level approaching to that at the beginning of pasture season. The pasture seasonal variations in the ratio were directly proportional to the corresponding content of CLA and indirectly proportional to the ratio in ewes’ milk fat. The results suggest that the seasonal variations in CLA content in ewes’ milk fat are related primarily to the seasonal variation in α-linolenic acid content in grass lipids.     

Pancreatic α-amylase and lipase inhibitory activity of polyphenolic compounds present in the extract of black chokeberry (Aronia melanocarpa L.)

The aim of this study was to investigate the effect of black chokeberry (Aronia melanocarpa L.) extract on the activity of porcine pancreatic α-amylase and lipase. An in vitro study demonstrated that three kinds of chokeberry extracts: methanolic, water and acetic caused inhibition of α-amylase and lipase. The methanolic and acetic extracts exhibited the highest inhibitory activities against α-amylase with the IC₅₀ values of 10.31 ± 0.04 mg/ml and pancreatic lipase 83.45 ± 0.50 mg/ml, respectively. In order to identify the compounds which may be the potential inhibitors of α-amylase and lipase, chokeberry extract was analyzed by preparative reverse phase chromatography and high performance liquid chromatography–mass spectrometry (HPLC–MS). These studies have shown that both anthocyanins and phenolic acids are compounds which inhibit the ability of the reaction catalyzed by α-amylase and lipase. The most effective inhibitor of pancreatic α-amylase was chlorogenic acid (IC₅₀ = 0.57 ± 0.16 mg/ml). In the group of anthocyanins the most potent inhibitor of α-amylase was cyanidin-3-glucoside (IC₅₀ = 1.74 ± 0.04 mg/ml), which also showed an ability to inhibit the reaction catalyzed by pancreatic lipase (IC₅₀ = 1.17 ± 0.05 mg/ml). These findings seem to indicate the use of chokeberry as a functional food component, contributing to its anti-obesity activities.     

Substrate effects on the mechanism of enantioselective hydrogenation using ruthenium bis(phosphine) complexes as catalyst: A mechanistic investigation of the hydrogenation of α,β-unsaturated acids and esters based on deuterium labeling studies

The mechanism of ruthenium-bis(phosphine) catalyzed enantioselective hydrogenation of olefins was examined using [Ru((R)-BINAP)(H)(MeCN)_n(sol)_3 − n]BF₄ (n = 0–3, sol = solvent used in reaction) as catalyst. Tiglic and angelic acids were used as standard α,β-unsaturated acid substrates; (Z)-methyl α-acetamidocinnamate and dimethyl itaconate were used as standard α,β-unsaturated ester substrates. Isotopic labeling studies (deuterium scrambling) indicate that two distinct mechanisms are in operation for α,β-unsaturated acids versus α,β-unsaturated esters. In each case, 5-membered metallocycle intermediates are formed via olefin-hydride insertion. The mechanisms, however, deviate primarily in the activation of dihydrogen, which is strongly affected by the nature of the substrate. Hydrogenation of α,β-unsaturated acids proceed via heterolytic cleavage of dihydrogen, whereas hydrogenation of α,β-unsaturated esters proceed via homolytic cleavage of dihydrogen. A full discussion of the mechanisms is presented.     

Inhibition of the activation of hepatic stellate cells by arundic acid via the induction of cytoglobin

BackgroundThe activation of hepatic stellate cells plays a central role in the development of liver fibrosis during chronic liver trauma. The aim of the present study was to identify a compound that inhibits the activation process of stellate cells.MethodsRat primary cultured stellate cells and a human stellate cell line (LX-2) were used. The effects of arundic acid on the expression of α-smooth muscle actin, collagen 1α1, and cytoglobin were evaluated.ResultsArundic acid (300 μM) inhibited the activation of primary rat stellate cells, as determined by morphological transformation and α-smooth muscle actin expression, after both prophylactic and therapeutic treatment. The level of α-smooth muscle actin mRNA showed a dose-dependent decrease in response to arundic acid, and 50 μM arundic acid exhibited the maximum inhibition of collagen 1α1 mRNA expression. In contrast, arundic acid triggered an unexpected increase in mRNA and protein levels of cytoglobin, the fourth globin in mammals expressed exclusively in hepatic stellate cells. The effect of arundic acid on the level of α-smooth muscle actin mRNA was abrogated in HSCs treated with cytoglobin siRNA. Arundic acid decreased the expression of collagen 1α1 mRNA in LX-2 cells.ConclusionArundic acid affects the activation process of hepatic stellate cells via the unexpected induction of cytoglobin.