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Trans-4-hydroxy-L-proline is an important amino acid that is widely used in medicinal and industrial applications, particularly as a valuable chiral building block for the organic synthesis of pharmaceuticals. Traditionally, trans-4-hydroxy-L-proline is produced by the acidic hydrolysis of collagen, but this process has serious drawbacks, such as low productivity, a complex process and heavy environmental pollution. Presently, trans-4-hydroxy-L-proline is mainly produced via fermentative production by microorganisms. Some recently published advances in metabolic engineering have been used to effectively construct microbial cell factories that have improved the trans-4-hydroxy-L-proline biosynthetic pathway. To probe the potential of microorganisms for trans-4-hydroxy-L-proline production, new strategies and tools must be proposed. In this review, we provide a comprehensive understanding of trans-4-hydroxy-L-proline, including its biosynthetic pathway, proline hydroxylases and production by metabolic engineering, with a focus on improving its production.

A new trans-4-hydroxy-l-proline (trans-Hyp) producing Bacillus cereus HBL-AI, was isolated from the air, which was screened just using l-proline as carbon and energy sources. This strain exhibited 73·4% bioconversion rate from initial l-proline (3 g l -1 ) to trans-Hyp. By sequencing the genome of this bacterium, 6244 coding sequences were obtained. Genome annotation analysis and functional expression were used to identify the proline-4-hydroxylase (BP4H) in HBL-AI. This enzyme belonged to a family of 2-oxoglutarate-related dioxygenases, which required 2-oxoglutarate and O 2 as co-substrates for the reaction. Homologous modelling indicated that the enzyme had two monomers and contained conserved motifs, which included a distorted 'jelly roll' β strand core and the residues (HXDXnH and RXS). The engineering Escherichia coli 3 Δ W3110/pTrc99a-proba-bp4h was constructed using BP4H, which transformed glucose to trans-Hyp in one step with high concentration of 46·2 g l -1 . This strategy provides a green and efficient method for synthesis of trans-Hyp and thus has a great potential in industrial application., (© 2020 The Society for Applied Microbiology.)

Hydroxyproline is an industrially important compound with applications in the pharmaceutical, nutrition, and cosmetic industries. trans-4-Hydroxy-L-proline is recognized as the most abundant of the eight possible isomers (hydroxy group at C-3 or C-4, cis- or trans-configuration, and L- or D-form). However, little attention has been paid to the rare isomers, probably due to their limited availability. This mini-review provides an overview of recent advances in microbial and enzymatic processes to develop practical production strategies for various hydroxyprolines. Here, we introduce three screening strategies, namely, activity-, sequence-, and metabolite-based approaches, allowing identification of diverse proline-hydroxylating enzymes with different product specificities. All naturally occurring hydroxyproline isomers can be produced by using suitable hydroxylases in a highly regio- and stereo-selective manner. Furthermore, crystal structures of relevant hydroxylases provide much insight into their functional roles. Since hydroxylases acting on free L-proline belong to the 2-oxoglutarate-dependent dioxygenase superfamily, cellular metabolism of Escherichia coli coupled with a hydroxylase is a valuable source of 2-oxoglutarate, which is indispensable as a co-substrate in L-proline hydroxylation. Further, microbial hydroxyproline 2-epimerase may serve as a highly adaptable tool to convert L-hydroxyproline into D-hydroxyproline. KEY POINTS: • Proline hydroxylases serve as powerful tools for selectivel-proline hydroxylation. • Engineered Escherichia coli are a robust platform for hydroxyproline production. • Hydroxyproline epimerase convertsl-hydroxyproline intod-hydroxyproline.

Trans-4-hydroxy-L-proline (Hyp) production by Escherichia coli (E. coli) in fermentation is a high-oxygen-demand process. E. coli secretes large amounts of soluble protein, especially in the anaphase of fermentation, which is an important factor leading to inadequate oxygen supply. And acetic acid that is the major by-product of Hyp production accumulates under low dissolved oxygen (DO). To increase DO and achieve high-level Hyp production, soluble protein was hydrolysed by adding protease in Hyp fermentation. The optimal protease, concentration, and addition time were trypsin, 0.2 g/L, and 18 h, respectively. With the addition of trypsin, the soluble protein in Hyp fermentation decreased by 43.5%. The DO could be maintained at 20-30% throughout fermentation. Hyp production and glucose conversion rate were 45.3 g/L and 18.1%, which were increases of 24.1% and 8.4%, respectively. The accumulation of acetic acid was decreased by 52.1%. The metabolic flux of Hyp was increased by 44.2% and the flux of acetate was decreased by 51.0%.

L-Hydroxyproline (Hyp) is a valuable intermediate for the synthesis of pharmaceuticals; consequently, a practical process for its production has been in high demand. To date, industrial processes have been developed by using L-Pro hydroxylases. However, a process for the synthesis of trans-3-Hyp has not yet been established, because of the lack of highly selective enzymes that can convert L-Pro to trans-3-Hyp. The present study was designed to develop a biocatalytic trans-3-Hyp production process. We speculated that ectoine hydroxylase (EctD), which is involved in the hydroxylation of the known compatible solute ectoine, may possess the ability to hydroxylate L-Pro, since the structures of ectoine and 5-hydroxyectoine resemble those of L-Pro and trans-3-Hyp, respectively. Consequently, we discovered that ectoine hydroxylases from Halomonas elongata, as well as some actinobacteria, catalyzed L-Pro hydroxylation to form trans-3-Hyp. Of these, ectoine hydroxylase from Streptomyces cattleya also utilized 3,4-dehydro-L-Pro, 2-methyl-L-Pro, and L-pipecolic acid as substrates. In the whole-cell bioconversion of L-Pro into trans-3-Hyp using Escherichia coli expressing the ectD gene from S. cattleya, only 12.4 mM trans-3-Hyp was produced from 30 mM L-Pro, suggesting a rapid depletion of 2-oxoglutarate, an essential component of enzyme activity as a cosubstrate, in the host. Therefore, the endogenous 2-oxoglutarate dehydrogenase gene was deleted. Using this deletion mutant as the host, trans-3-Hyp production was enhanced up to 26.8 mM from 30 mM L-Pro, with minimal loss of 2-oxoglutarate. This finding is not only beneficial for trans-3-Hyp production, but also for other E. coli bioconversion processes involving 2-oxoglutarate-utilizing enzymes.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and high mortality, posing a major threat to human health. Increased levels of inflammatory cytokines, reactive oxygen species and coagulation cascade have been extensively reported in IPF. We previously fused Hirudin and human manganese superoxide dismutase (hSOD2) to generate a dual-feature fusion protein, denoted as rhSOD2-Hirudin fusion protein. In this study, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and Hydroxyproline (HYP) assays were used to investigate the effects of rhSOD2-Hirudin protein on thrombin-induced fibroblast proliferation and collagen accumulation in vitro. Subsequently, the mice model of pulmonary fibrosis induced by bleomycin was used for evaluating the anti-inflammatory and anti-fibrotic effects of rhSOD2-Hirudin protein in vivo. Results showed that rhSOD2-Hirudin protein could inhibit the proliferation of fibroblasts and reduce the HYP production in vitro by inhibiting the activity of thrombin. In vivo experiments showed that lung inflammation and fibrosis were significantly decreased in rhSOD2-Hirudin protein-treated mice. Furthermore, rhSOD2-Hirudin protein treatment reduced profibrotic protein and gene expression while reducing the number of inflammatory cells in the lung. In conclusion, rhSOD2-Hirudin protein can effectively attenuate pulmonary fibrosis in vitro and in vivo, mainly by inhibiting the activity of thrombin meanwhile increasing SOD2 levels prevent cells from being damaged by reactive oxygen species, thereby mitigating IPF progression. This study provided important information on the feasibility and efficacy of rhSOD2-Hirudin protein as a novel therapeutic agent for IPF., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Nickel oxide nanoparticles (Nano NiO) could induce pulmonary fibrosis, however, the mechanisms are still unknown. The aim of the present study was to explore the roles of transforming growth factor-β1 (TGF-β1), mitogen-activated protein kinase (MAPK) pathway and MMPs/TIMPs balance in Nano NiO-induced pulmonary fibrosis. For that purpose, we first established Nano NiO-induced human lung adenocarcinoma epithelial cells (A549 cells) model of collagen excessive formation through detecting the levels of hydroxyproline (Hyp) and type I collagen (Col-I). Then the protein levels of TGF-β1, MAPKs, and MMPs/TIMPs were assessed by Western blot. The results showed that Nano NiO resulted in the increased contents of Hyp, Col-I, and TGF-β1, the MAPK pathway activation and MMPs/TIMPs imbalance with a dose-dependent manner. In addition, to investigate whether TGF-β1 mediated MAPK signaling pathway, A549 cells were treated by 100 μg/mL Nano NiO combined with TGF-β1, p38 MAPK, and ERK1/2 inhibitors (10 μM SB431542, 10 μM SB203580, and 10 μM U0126), respectively. We found that MAPK signal pathway was suppressed by TGF-β1 inhibitor. Meanwhile, the increased contents of Hyp and Col-I, and MMPs/TIMPs imbalance were alleviated by the p38 MAPK and ERK1/2 inhibitors, respectively. These findings indicated that the MAPK pathway and MMPs/TIMPs imbalance were involved in collagen excessive formation induced by Nano NiO., (© 2019 Wiley Periodicals, Inc.)

trans-4-Hydroxy-l-proline (trans-4Hyp) is widely used as a valuable building block for the organic synthesis of many pharmaceuticals such as carbapenem antibiotics. The major limitation for industrial bioproduction of trans-4Hyp is the low titer and productivity by using the existing trans-proline 4-hydroxylases (trans-P4Hs). Herein, three new trans-P4Hs from Alteromonas mediterranea (AlP4H), Micromonospora sp. CNB394 (MiP4H) and Sorangium cellulosum (ScP4H) were discovered through genome mining and enzymatic determination. These trans-P4Hs were introduced into an l-proline-producing chassis cell, and the recombinant strain overexpressing AlP4H produced the highest concentration of trans-4Hyp (3.57 g/L) from glucose in a shake flask. In a fed-batch fermentation with a 5 L bioreactor, the best strain SEcH (pTc-B74A-alp4h) accumulated 45.83 g/L of trans-4Hyp within 36 h, with the highest productivity (1.27 g/L/h) in trans-4Hyp fermentation from glucose, to the best of our knowledge. This study provides a promising hydroxylase candidate for efficient industrial production of trans-4Hyp., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Bioconversion of l-proline using recombinant strain with proline-4-hydroxylase (P4H) is a preferred biocatalytic process in the economical production of trans-Hyp. In this study, a recombinant E. coli overexpressing hydroxylase (P4H), γ-glutamyl kinase and glutamate-semialdehyde dehydrogenase (ProBA) genes were constructed by knocking out the key genes in the metabolism. These key genes contained putA encoding proline dehydrogenase (PutA) in the l-proline metabolism and other catalytic enzyme genes, sucAB encoding α-ketoglutarate dehydrogenase (SucAB), aceAK encoding isocitratelyase (AceA) and isocitrate dehydrogenase kinase/phosphatase (AceK) in the TCA cycle. This recombinant strain coupled the synthetic pathway of trans-Hyp with TCA cycle of the host strain. It inhibited the consumption of l-proline completely and promoted the accumulation of 2-oxoglutarate (2-OG) as a co-substrate, which realized the highest conversion of glucose to trans-Hyp. A fed-batch strategy was designed, capable of producing 31·0 g l -1 trans-Hyp from glucose. It provided a theoretical basis for commercial conversion of glucose to trans-Hyp., Significance and Impact of the Study: Trans-4-Hydroxy-l-proline (trans-Hyp) is a valuable chiral building block for the synthesis of pharmaceutical intermediates. Unsatisfactory microbial bioconversion resulted in a low yield of trans-Hyp. In this study, we blocked the unwanted blunting pathways of host strain and make the cell growth couple with the trans-Hyp synthesis from glucose. Finally, a recombinant Escherichia coli with short-cut and efficient trans-Hyp biosynthetic pathway was obtained. It provided a theoretical basis for commercial production of trans-Hyp., (© 2018 The Society for Applied Microbiology.)

Radiation-induced lung injury has restricted radiotherapy for thoracic cancer. The purpose of this study was to investigate the radioprotective effects of bromodomain and extra terminal (BET) inhibitor JQ1 in a murine model of pulmonary damage. Chest computed tomography (CT) was performed in a rat model after 20 Gy radiation of the right thorax. And histological evaluation and protein expressions of irradiated tissue were analyzed to confirm the potential anti-fibrosis effect of JQ1 and its underlying mechanisms. Moreover, colony formation assays were used to explore the effects of JQ1 on esophageal cancer Eca109 and breast cancer MCF7. JQ1 attenuated radiologic and histologic presentations of radiation-induced fibrosis, inflammatory reaction and pulmonary structural changes and the increase of Hounsfield units (HU) density and hydroxyproline content after radiation. Additionally, JQ1 suppressed BRD4, c-MYC, Collagen I, TGF-β, p-NF-κB p65, p-Smad2 and p-Smad3 expressions after irradiation, repressed proliferation and transdifferentiation of lung fibroblasts, and impaired clonogenic survival of thoracic cancer cells. Collectively, our study demonstrated for the first time that BET Bromodomain inhibitor JQ1 protected normal lung tissue after radiation, and exerted a radiosensitizing effect in thoracic cancer cells.

Glycine, proline, and hydroxyproline (Hyp) contribute to 57% of total amino acids (AAs) in collagen, which accounts for one-third of proteins in animals. As the most abundant protein in the body, collagen is essential to maintain the normal structure and strength of connective tissue, such as bones, skin, cartilage, and blood vessels. Mammals, birds, and fish can synthesize: (1) glycine from threonine, serine, choline, and Hyp; (2) proline from arginine; and (3) Hyp from proline residues in collagen, in a cell- and tissue-specific manner. In addition, livestock (e.g., pigs, cattle, and sheep) produces proline from glutamine and glutamate in the small intestine, but this pathway is absent from birds and possibly most fish species. Results of the recent studies indicate that endogenous synthesis of glycine, proline, and Hyp is inadequate for maximal growth, collagen production, or feed efficiency in pigs, chickens, and fish. Although glycine, proline and Hyp, and gelatin can be used as feed additives in animal diets, these ingredients except for glycine are relatively expensive, which precludes their inclusion in practical rations. Alternatively, hydrolyzed feather meal (HFM), which contains 9% glycine, 5% Hyp, and 12% proline, holds great promise as a low cost but abundant dietary source of glycine, Hyp, and proline for ruminants and nonruminants. Because HFM is deficient in most AAs, future research efforts should be directed at improving the bioavailability of its AAs and the balance of AAs in HFM-supplemented diets. Finally, HFM may be used as a feed additive to prevent or ameliorate connective tissue disorders in domestic and aquatic animals.

Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase β subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na + /l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Fibrosis is the excessive accumulation of extracellular matrix components due to chronic injury, with collagens as predominant structural components. Liver fibrosis can progress to cirrhosis, which is characterized by a severe distortion of the delicate hepatic vascular architecture, the shunting of the blood supply away from hepatocytes and the resultant functional liver failure. Cirrhosis is associated with a highly increased morbidity and mortality and represents the major hard endpoint in clinical studies of chronic liver diseases. Moreover, cirrhosis is a strong cofactor of primary liver cancer. In vivo models are indispensable tools to study the cellular and molecular mechanisms of liver fibrosis and to develop specific antifibrotic therapies towards clinical translation. Here, we provide a detailed description of select optimized mouse models of liver fibrosis and state-of-the-art fibrosis readouts.

trans-4-Hydroxy-l-proline (Hyp) is a chiral amino acid conventionally produced by acid hydrolysis of animal collagen, a process which involves the bottleneck problems of low efficiency and heavy environmental pollution. Biotransformation of l-proline into Hyp using recombinant whole-cell biocatalysis with proline-4-hydroxylase (P4H) is an environmentally-friendly alternative method. Since biohydroxylation of proline by whole cells is a high-oxygen-demand process, oxygen transfer needs to be improved. To solve this problem, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosome of recombinant Escherichia coli expressing the P4H gene originally from Dactylosporangium sp. RH1. Expression of Vitreoscilla hemoglobin (VHb) resulted in a 94.4% increase of Hyp production in a 100-mL shaking flask culture compared to the same strain without VHb expression. Meanwhile in a fed-batch fermentation with a 1.4 L bioreactor, the expression of VHb led to an increase in Hyp production by 73.2% and biomass improved by 106%. We also found that acetic acid concentration was decreased by the expression of VHb during the fermentation. This work demonstrates that vgb chromosomal integration is an efficient way to improve Hyp production by enhancing oxygen transfer in recombinant E. coli., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Background & Aims: Bone mineral loss, reduced lung function and impaired nutritional status are frequently present in children with Cystic Fibrosis (CF). Blood concentrations and urinary excretion of hydroxyproline (OH-PRO) have been used as markers of bone mineral status and lung function in CF., Objective: To examine whether whole body hydroxyproline production, as assessed by a new stable isotope methodology, is increased in pediatric patients with CF and associated with bone mineral loss, lung function decline and impaired nutritional status., Design: In a cross-sectional study in 15 pediatric patients with CF and 17 healthy young control subjects, whole body hydroxyproline production (Wb OH-PRO) was assessed in the postabsorptive state by primed-constant and continuous infusion of the stable isotope 2-D-OH-PRO for 3 h. Bone mineral density (BMD) of whole body, hip and spin, and body composition (fat mass and fat-free mass) were determined by dual-energy X-ray Absorptiometry (DXA). Plasma isotopic enrichments and OH-PRO concentrations were measured by LC/MS/MS., Results: Higher values for WbOH-PRO production and plasma OH-PRO concentrations were found in pediatric CF patients than in the healthy young subjects (p < 0.001). WbOH-PRO production was significantly correlated with plasma OH-PRO concentrations in the CF (r: 0.70, p = 0.007) but not in the healthy group. WbOH-PRO production in CF was associated with low BMD values in hip (r = -0.61, p = 0.02) and spine (r = -0.59, p = 0.02) but not with whole body BMD, lung function or body composition., Conclusion: A new stable isotope approach revealed enhanced levels of whole body hydroxyproline production rate in pediatric patients with CF, indicative of enhanced whole body collagen breakdown. Increased levels for whole body hydroxyproline production in CF were associated with severe bone mineral loss in hip and spine but not with lung function decline or impaired nutritional status. Registration ClinicalTrials.gov = NCT01172301., (Copyright © 2013 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Background: Trans-4-hydroxy-L-proline (trans-Hyp), one of the hydroxyproline (Hyp) isomers, is a useful chiral building block in the production of many pharmaceuticals. Although there are some natural biosynthetic pathways of trans-Hyp existing in microorganisms, the yield is still too low to be scaled up for industrial applications. Until now the production of trans-Hyp is mainly from the acid hydrolysis of collagen. Due to the increasing environmental concerns on those severe chemical processes and complicated downstream separation, it is essential to explore some environment-friendly processes such as constructing new recombinant strains to develop efficient process for trans-Hyp production., Result: In this study, the genes of trans-proline 4-hydroxylase (trans-P4H) from diverse resources were cloned and expressed in Corynebacterium glutamicum and Escherichia coli, respectively. The trans-Hyp production by these recombinant strains was investigated. The results showed that all the genes from different resources had been expressed actively. Both the recombinant C. glutamicum and E. coli strains could produce trans-Hyp in the absence of proline and 2-oxoglutarate., Conclusions: The whole cell microbial systems for trans-Hyp production have been successfully constructed by introducing trans-P4H into C. glutamicum and E. coli. Although the highest yield was obtained in recombinant E. coli, using recombinant C. glutamicum strains to produce trans-Hyp was a new attempt.

Background: Delayed wound healing is considered one of the most serious diabetes-associated complications. The presence of replicating organisms such as bacteria within a diabetic's wound is considered one of the most important factors that impair cutaneous wound healing and the potential cellular and/or molecular mechanisms that are involved in the healing process. Defensins, which are anti-microbial peptides, have potent bactericidal activity against a wide spectrum of the bacterial and fungal organisms that are commonly responsible for wound infections. We recently demonstrated that camel whey proteins (WPs) expedite the healing of diabetic wounds by enhancing the immune response of wounded tissue cells and by alleviating some of the diabetic complications., Methods: In the present study, we investigated the effects of WP supplementation on the mRNA and protein expression levels of β-defensin-1 (BD-1), 2 and 3 and subsequently on the wound healing process in a streptozotocin (STZ)-induced diabetic mouse model. In this study, three groups of mice were used (10 mice per group): group 1, the non-diabetic mice (control); group 2, the diabetic mice; and group 3, the diabetic mice that received a daily supplement of undenatured WP (100 mg/kg of body weight) via oral gavage for 1 month., Results: Compared with the non-diabetic control mice, the diabetic mice exhibited delayed wound closure that was characterized by a reduction in hydroxyproline content (indicator of collagen deposition), a marked elevation in free radical levels and a prolonged elevation in the levels of inflammatory cytokines, including interleukin-6 (IL-6), transforming growth factor-beta (TGF-β) and tumor necrosis factor-alpha (TNF-α). Interestingly, compared with the diabetic mice that did not receive WP supplementation, the diabetic mice with WP had an accelerated closure and healing process of their wounds. The WP supplementation also decreased their levels of free radicals and restored their hydroxyproline content; proinflammatory cytokine levels; and expression of BD-1, 2 and 3 in the wounded tissue., Conclusion: WP supplementation may be beneficial for improving the healing and closure of diabetic wounds.

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α(2)β(2) tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I-III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.

Pulmonary fibrosis (PF) is a progressive fatal disorder. Bleomycin (BLM) is a widely used chemotherapeutic agent causing PF. Numerous agents have been investigated to prevent the progression of PF so far, but there is still a need to find more efficacious agents. Proanthocyanidin (PA) is a strong antioxidant, the main ingredient of grape seed extract. Since PA is ready for use in practice, we aimed to compare the preventive effect of PA in comparison with taurine (Tau) in BLM-induced PF. Forty Wistar male albino rats were used in the study and were divided into four groups: group 1, control; group 2, BLM-induced PF group; group 3, BLM-induced PF and treated with PA group; and group 4, BLM-induced PF and treated with Tau group. Treatments were begun 10 days before and continued 21 days after BLM injection. PA and Tau effectively inhibited inflammation, edema, severity of fibrosis, fibrosis extension, inflammatory cell accumulation, iNOS staining, and hydroxyproline level as well (p < 0.05). Total histological scores of the PA group were similar to the control group; Tau was significantly higher than the control group but lower than the BLM group (p < 0.05). We believe that PA could be a new treatment choice for PF, but further studies need to be conducted to verify the findings of the current study.

The proline analog cis-4-hydroxy-L-proline (CHOP), which inhibits the biosynthesis of collagen, has been evaluated as an anticancer, antifibrosis, and antihypertension drug. However, its water solubility and low molecular weight limit its therapeutic potential since it is rapidly excreted. In addition, CHOP is considered to be too toxic due primarily to its systematic effects on noncollagen proteins. To promote retention in blood or decrease toxicity, N-acetylation of CHOP might be a novel approach as a prodrug, instead of other approaches such as the conjugation of poly(ethylene glycol-Lys) or the modification of O-acetylation. In this study, we found that N-acetyltransferase Mpr1 that detoxifies the proline analog azetidine-2-carboxylate in Saccharomyces cerevisiae also converts CHOP into N-acetyl CHOP in vitro and in vivo. Escherichia coli BL21(DE3) cells overexpressing Mpr1 showed greater CHOP resistance than those carrying the vector. To increase the productivity of N-acetyl CHOP, the addition of NaCl into the medium that induces osmotic stress accelerates CHOP uptake into E. coli cells. As a result, the amount of N-acetyl CHOP production in Mpr1-overexpressing cells was 3.5-fold higher than that observed in the cells cultured in the absence of NaCl. The highest yield was achieved during the exponential growth phase of cells in the presence of 2% NaCl (52 μmol N-acetyl CHOP per g wet cell weight). Our results provide a promising approach to microbial production of N-acetyl CHOP as a new prodrug., (Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This study was designed to investigate the hepatoprotective effects of magnesium chenoursodeoxycholic acid (Mg-CUD), a magnesium trihydrate salt of ursodeoxycholic acid and chenodeoxycholic acid, in carbon tetrachloride (CCl(4))-induced liver fibrosis in rats. Rats were treated with CCl(4) dissolved in olive oil (0.5 mL/kg, twice a week) intraperitoneally for eight weeks. Mg-CUD was administered orally at 15.625, 31.25, 62.5 and 125 mg/kg once a day. Chronic CCl(4) administration induced increases in serum transforming growth factor-β1, hepatic hydroxyproline content and serum alanine aminotransferase activity. Mg-CUD attenuated these increases. The levels of α-smooth muscle actin protein and mRNA expression were increased by chronic CCl(4) exposure and Mg-CUD attenuated these increases. Mg-CUD suppressed increases in matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 mRNA expression and elevation of oxidative stresses by attenuating lipid peroxidation and enhancing reduced glutathione/oxidized glutathione ratio. The overexpression of toll-like receptor 4 and increased nuclear translocation of nuclear factor-κB and phosphorylated c-Jun, a component of activator protein 1, were suppressed by Mg-CUD. Furthermore, CCl(4) increased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and cyclooxygenase (COX)-2. Mg-CUD attenuated the levels of TNF-α, IL-6 and COX-2, while it augmented the level of IL-10. Our results suggest that Mg-CUD may prevent liver fibrosis by modulating collagen accumulation and inflammatory signaling pathways.

Aim: To investigate the influence of remote ischemic preconditioning (RIPC) on anastomotic integrity., Methods: Sixty male Wistar rats were randomized to six groups. The control group (n = 10) had an end-to-end ileal anastomosis without RIPC. The preconditioned groups (n = 34) varied in time of ischemia and time of reperfusion. One group received the amino acid L-arginine before constructing the anastomosis (n = 9). On postoperative day 4, the rats were re-laparotomized, and bursting pressure, hydroxyproline concentration, intra-abdominal adhesions, and a histological score concerning the mucosal ischemic injury were collected. The data are given as median (range)., Results: On postoperative day 4, median bursting pressure was 124 mmHg (60-146 mmHg) in the control group. The experimental groups did not show a statistically significant difference (P > 0.05). Regarding the hydroxyproline concentration, we did not find any significant variation in the experimental groups. We detected significantly less mucosal injury in the RIPC groups. Furthermore, we assessed more extensive intra-abdominal adhesions in the preconditioned groups than in the control group., Conclusion: RIPC directly before performing small bowel anastomosis does not affect anastomotic stability in the early period, as seen in ischemic preconditioning.

Background: Airway remodeling is one of the cardinal features of asthma and is thought to play a pivotal role in refractory or persistent asthma. Immunoglobulin E (IgE) has a major effect on the pathogenesis of asthma. The aim of this study was to investigate the effects of anti-IgE antibody not only on airway inflammation and bronchial hyperresponsiveness, but also on airway remodeling in a murine model of chronic asthma., Methods: The authors developed a mouse model of chronic asthma in which ovalbumin (OVA)-sensitized female BALB/c-mice were exposed to intranasal OVA administration twice a week for 3 months. Anti-IgE antibodies were administered intravenously starting on the 38th day and once a month thereafter for 3 months during the intranasal OVA challenge., Results: Mice that were chronically exposed to OVA developed sustained eosinophilic airway inflammation and airway hyperresponsiveness (AHR) to methacholine and showed increased levels of collagen, hydroxyproline, and alpha-smooth muscle actin, as compared with control mice. Treatment with anti-IgE antibody inhibited the development of AHR, eosinophilic inflammation, and airway remodeling. Moreover, anti-IgE antibody treatment reduced the levels of interleukin (IL)-5 and IL-13 in the bronchoalveolar lavage fluids, although it did not affect the levels of IL-10, transforming growth factor-beta, and activin A., Conclusion: These results suggest that anti-IgE antibody treatment modulates the airway inflammation and remodeling associated with chronic allergen challenge. The inhibition of inflammation may be related to the regulation of Th2 cytokines. However, the mechanisms underlying the blocking of airway remodeling by anti-IgE antibody remain to be elucidated.

Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on-off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein-protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor alpha, which plays a key role in sensing molecular oxygen, and could act on inhibitory kappaB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification.

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing L-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-L-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-L-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel L-proline cis-4-hydroxylases converting free L-proline to cis-4-hydroxy-L-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected L-proline cis-4-hydroxylase activity in both proteins. Likewise L-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).

Objectives: To evaluate the efficacy in vitro and in vivo of a new antibacterial suture (PGAB) compared with a traditional braided suture (PG). Our primary goals were to study microbiological effectiveness and impact on wound healing of PGAB vs PG. Secondary goal was to analyze influence on inflammatory response., Methods: In vitro study: clinical samples of Staphylococcus epidermidis, Staphylococcus aureus, S. hominis, Staphylococcus haemolyticus, Staphylococcus auricularis, Enterococcus faecalis, Corynebacterium spp. and Escherichia coli were studied. We also implanted a flat mesh in 10 minipigs, four incisions each (two PG and two PGAB) two contaminated with S. epidermidis and two not contaminated. Finally, we performed four colic anastomosis in each of 10 minipigs, two contaminated with E. coli and two not contaminated (two PG and two PGAB). We studied the inflammatory and wound healing processes in both models., Results: We observed a bactericidal efficacy of PGAB against grampositive, and bacteriostatic effect against E. coli. Mesh study: recovered CFU were lower in the group PGAB vs PG. In the group PGAB, inflammatory mediators' concentrations were lower. In the group PGAB, concentrations of wound healing mediators were normal. Colic anastomosis: recovered CFU were lower in the group PGAB vs the group PG. In the group PGAB we observed a reduction of inflammatory mediators. In the group PGAB we observed normalized concentrations of wound healing mediators., Conclusions: This study demonstrates microbiological efficacy of PGAB, that normalizes wound healing process, and an anti-inflammatory effect.

The goal of the study is to investigate the effects of the 1,320-nm Nd:YAG laser on skin barrier function and dermal remodeling. Forty-eight female mice were irradiated with the 1,320-nm laser four times at an interval of 1 week (350 micros, 10 mm, single pass 10% overlap). Three doses were used: 18, 22, and 26 J/cm2. Transepidermal water loss (TEWL) was measured at day 0, 1, 7, 21, 30, and 60 after treatments. Skin biopsies were taken to observe the changes in dermal structures and quantify the increases in the number of fibroblasts and collagen content. The dose of 22 J/cm2 induced the lowest ratio of TEWL (0.58+/-0.06, p < 0.05), highest degree of fibroblast proliferation (45.68+/-5.70, p < 0.05), and the most increase in type I collagen fibers (49.8%, p < 0.05) and hydroxyproline content (85.7%, p < 0.05). The dose of 26 J/cm2 caused marked inflammatory infiltration and the most increase in type III collagen fibers. The increase of type I collagen fibers was greater than type III collagen fibers for all three doses (p < 0.05). Our results demonstrated that 22 J/cm2 was most effective in enhancing skin barrier function and stimulating new collagen synthesis. Dermal remodeling after the 1,320-nm laser was mainly through the synthesis and deposition of collagen type I. Inflammatory reactions were in favor of the formation of type III collagen.

Cigarette smokers deposit less collagen, expressed as hydroxyproline, in granulation tissue than nonsmokers. We studied the effect of abstinence from smoking and transdermal nicotine patches on deposition of hydroxyproline, proline, type I procollagen, and total proteins. Fifty-four healthy smokers were studied during 10 days of smoking and again from days 10 to 20 following smoking cessation. After the first 10 days of abstinence they were randomized to double-blind treatment with transdermal nicotine patches of 25 mg/day or placebo for a period of 10 days. During this period and during smoking, an expanded polytetrafluoroethylene tube was implanted into the subcutis. Following removal of the implant, total amino acids and peptides were extracted. Hydroxyproline and proline were analyzed by high-pressure liquid chromatography, type I procollagen was analyzed by enzyme-linked immunoassay, and total proteins were determined colorimetrically. In the 39 subjects who complied with the study protocol, abstinence from smoking did not affect the deposition of hydroxyproline, proline, type I procollagen, or total protein in the implants. During abstinence, the type I procollagen level increased by 18% in the transdermal nicotine patches group and decreased by 10% in the placebo group (p<0.05). We conclude that 20 days of abstinence from smoking does not affect collagen deposition in granulation tissue. However, in abstinent smokers, transdermal nicotine patches appears to increase type I collagen synthesis.

Objective: To investigate the in vitro effects of dehydroepiandrosterone (DHEA) on neonatal rat chondrocytes., Design: Chondrocytes isolated from neonatal rat cartilage were cultured in three-dimensionally agarose beads and were treated with DHEA., Methods: Primary culture of chondrocytes was harvested from newborn Wistar rats. The DHEA effects on chondrocyte activities were evaluated by analyzing chondrocyte proliferation, matrix protein synthesis, gene expressions of collagen, matrix metalloproteinase-1, -3 and -13 (MMP-1, -3 and -13), and cyclooxygenase-2 (COX-II), and protein synthesis of interleukin-6 (IL-6), prostaglandin E2 (PGE2) and tissue inhibitor of metalloproteinase-1 (TIMP-1)., Results: The DHEA treatment did affect chondrocyte proliferation and glycosaminoglycan (GAG) synthesis. DHEA suppressed the expression of MMP-1, -3 and -13 genes and PGE2 protein synthesis enhanced by lipopolysaccharide (LPS) while the COX-II and inducible nitric oxide synthase (iNOS) gene expressions were down-regulated by DHEA., Conclusions: Our study demonstrates that DHEA has an ability to modulate the imbalance between MMPs and PGE2 in the neonatal chondrocytes which suggest that it has a potential protective role against articular cartilage damage.

Background and Aims: Proton pump inhibitors (PPI) and prostaglandin (PG) preparations are believed to both prevent NSAID-induced gastric ulcers and promote the delayed healing of gastric ulcers by NSAIDs, but it remains unclear which of these drugs is superior. The aim of this study was to clarify which achieved better healing of NSAID-induced gastric ulcers, not only with respect to epithelialization but also repair of the submucosal tissues., Methods: We used acetic acid to induce gastric ulcers in rats, and compared the changes between a control group, NSAID group, NSAID + PPI group and NSAID + PG group. After removing the stomach of each animal, an ulcer index was calculated and the collagen content and type III collagen content of granulation tissue were measured. We also studied fibroblast dynamics, including proliferation, collagen synthesis, differentiation into myofibroblasts, and apoptosis., Results: Indomethacin prevented re-epithelialization of the ulcers, interfered with fibroblast function, and also delayed the replacement of type III collagen. Omeprazole promoted epithelialization, but could not fully reverse the influence of indomethacin on granulation tissue maturation. A concomitant dose with misoprostolreversed it completely., Conclusions: From our point of view in this study in the use of experimental ulcers, it was thought that compensation of PG should have priority to gastric acid inhibition in terms of healing of NSAID-induced gastric ulcer., (Copyright (c) 2006 S. Karger AG, Basel.)

A bioreactor system consisting of a multifunctional stimulation unit and common 6-well culture plate is introduced to activate extracellular matrix synthesis in intervertebral disc cells due to cyclic mechanical strain. The developed stimulation unit is sterilizable and reusable. It is viable for cultivation and mechanical stimulation of cartilage tissue and tissue engineered cell matrix constructs in combination with the common 6-well culture plate. The custom made device allows long-term cultivations in batch- or continuous operation mode. Manual handling and thereby the risk of contamination is reduced. Sampling, changing the medium, and addition of supplements are easily performed from the connected conditioning vessel. This bioreactor system enables stimulation of different samples independently during one run. For the work presented here anulus fibrosus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs. Over a period of 14 days the constructs were subjected to 10% compression under cyclic mechanical pressure with a frequency of 0.1 Hz. Afterwards the constructs were biochemically examined for hydroxyproline and sulphated glycosaminoglycanes. These proven constituents of extracellular matrix were found to be released depending on the applied compressive strain.

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors.Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes. There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism.

This study presents the antifibrotic effect of interferon gamma (IFN-gamma) in silicosis of rats. Wistar rats were received saline or 20 mg of quartz by intratracheal instillation. The next day after instillation, silicosis model of rats were daily treated IFN-gamma (1.0 x 10(5) units/rat) or saline by intramuscular route. At 1 month or 2 months after instillation, rats were sacrificed to determine hydroxyproline content and observe the change of fibrosis and expression of interleukin-4 (IL-4) and transforming growth factor-beta1 (TGF-beta1) in the lung of rats. The hydroxyproline content of IFN-gamma treated groups was significantly lower than that of quartz groups (P<0.05). At 1 month after instillation, there were Stage II and II+ of silicotic nodules in quartz group; however, in IFN-gamma treated group only cellular nodules (Stage I) were observed. At 2 months after instillation, there was Stage II+ and III of silicotic nodules in quartz group. In IFN-gamma treated group Stage II was found. The expressions of IL-4 (2.19+/-0.90, 0.61+/-0.22, respectively) and TGF-beta1 (1.37+/-0.31, 1.76+/-0.72, respectively) in IFN-gamma treated groups were significantly lower than those in quartz groups (IL-4: 3.33+/-1.27, 1.99+/-0.80; TGF-beta1: 3.67+/-0.63, 4.90+/-1.11, respectively; P<0.05 or P<0.01). It may be concluded that the decreased expression of IL-4 and TGF-beta1 may be associated with decreased hydroxyproline synthesis, which then results in decreased lung fibrosis.

Background & Aims: One of the most frequent complications in patients with cancer and malnutrition is the surgical wound healing delay or failure. Some studies have shown that arginine improves wound healing in rodents and in healthy human beings. The main objective of this study was to assess the effect of early postoperative enteral immunonutrition on the wound healing process in patients undergoing surgery for gastric cancer., Methods: Sixty six patients with gastric cancer were randomized to receive early postoperative enteral immunonutrition (formula supplemented with arginine, omega-3 fatty acids and ribonucleic acid (RNA)) or an isocaloric-isonitrogenous control. Assessment of wound healing process: (1) Quantification of hydroxyproline deposition in a subcutaneously placed catheter, (2) occurrence of surgical wound healing complications., Results: Sixty patients were analyzed. Patients fed with immunonutrition (n=30) showed higher local hydroxyproline levels (59.7 nmol (5.0-201.8), vs. 28.0 nmol (5.8-89.6) P=0.0018) and significantly lower episodes of surgical wound healing complications (0 vs. 8 (26.7%) P=0.005) when compared to patients fed with the control formula (n=30)., Conclusions: Early postoperative enteral nutrition with a formula supplemented with arginine, omega 3 fatty acids and RNA increased hydroxyproline synthesis and improved surgical wound healing in patients undergoing gastrectomy for gastric cancer.

Repair of degenerated intervertebral discs by engineered tissue is a clinical challenge in spinal surgery. Prerequisites are cultivation of intervertebral disc cells and determination of their biologic properties. The influence of disc damage in different spinal disorders on the outcome of disc cell cultures has not been discussed previously. This study showed the feasibility of cultivation of cells from damaged human intervertebral discs and the dependence of cellular culture properties on the underlying disc disorder. Human intervertebral disc cells were isolated from disc tissue obtained during surgical procedures for scoliosis, osteochondrosis, and disc herniation. After proliferation in monolayer culture, cells were embedded in a mixed matrix composed of fibrin and hyaluronic acid. Deoxyribonucleic acid content, hydroxyproline content, and proteoglycan synthesis were determined on Days 7, 14, and 21. In a three-dimensional environment only cells obtained from scoliotic and osteochondrotic discs showed significant deoxyribonucleic acid and proteoglycan synthesis. However, hydroxyproline content increased only in cells from scoliotic discs. The results of this study show that the formation of extracellular matrix components under three-dimensional culture conditions is dependent on the nature of intervertebral disc damage of the tissue processed.

Hypoalbuminemia is often claimed to impair wound healing, and therefore albumin has traditionally been administered to derive beneficial effects on general physiologic conditions including the nutritional state. However, the influence of albumin administration on systemic protein metabolism and wound healing is still unclear. Therefore, the objective of this study was to investigate the influence of albumin administration on protein metabolism and wound healing in burned rats. After receiving basic total parenteral nutrition (TPN) for 4 days, Sprague-Dawley rats underwent a 6-cm skin incision in the back and a burn involving 20 percent of the whole body surface. The rats were divided into three groups. Group I continued to receive basic TPN. Group II was given basic TPN, but 20 percent of the total nitrogen was replaced by albumin. Group III was administered basic TPN plus albumin equivalent to 20 percent of the total nitrogen of basic TPN. Group IV had the skin incision but no burn, receiving only basic TPN. All the groups were euthanized 4 days after the burn or skin incision. The wound healing potential in terms of tensile strength was enhanced by replacement and addition of albumin (groups II and III, respectively) after a 20 percent burn. Hydroxyproline levels in the wound tended to increase in group II, and significantly increased in group III. Whereas albumin replacement (group II) did not remarkably change the protein metabolism, albumin addition (group III) significantly increased both protein synthesis (S) and breakdown (B) with the S/B ratio and nitrogen balance remaining the same as with albumin-free nutrition (group I). The urinary 3-methyl-histidine/creatinine ratio significantly increased after burn in group III. We conclude that intravenous albumin administration enhanced incisional wound healing in burned rats. Increased protein synthesis with concurrent myolysis and protein breakdown by albumin addition (group III) was observed during wound healing.

Objective: The defects of collagen metabolism are responsible for the disorganization of extracellular matrix in stomach cancer. Collagen through interaction with integrin receptors regulates the cellular growth, differentiation, gene expression, prolidase and gelatinase activity and plays an important role in tumorigenesis and invasiveness. Although extracellular metalloproteinases initiate the breakdown of collagen in tissues, the final step of its degradation is mediated by prolidase. Therefore, we decided to compare the degradation of collagen in control tissues to gastric cancer tissues., Methods: We investigated the collagen content (hydroxyproline assay), expressions of beta(1) integrin, prolidase and gelatinases A and B (Western immunoblot) as well activities of prolidase (colorimetric assay) and gelatinases (zymography) in stomach cancer tissue (n = 10). The results were compared with corresponding data obtained for control tissues (n = 10)., Results: No differences in the collagen content were found between the studied tissue samples. However, an increase in free proline pool, enhanced gelatinase expression and elevated gelatinolytic activity were found in the tumor tissue. These phenomena were accompanied by a significant elevation in prolidase activity and an increase in beta(1) integrin expression in stomach cancer, compared to control tissue., Conclusion: The data presented suggest an enhancement of collagen turnover in stomach cancer. It may be suggested that the increased degradation of collagen by gelatinase in cancer tissue is balanced by an increased biosynthesis of this protein., (Copyright 2004 S. Karger AG, Basel.)

Purpose: Extracts of the herb Tripterygium wilfordii hook f, the major component of which is triptolide, have been used in traditional Chinese medicine for the treatment of rheumatoid arthritis. Triptolide also exerts many other biological actions both in vitro and in vivo. The effect of this agent on collagen degradation by cultured corneal fibroblasts was examined., Methods: Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen and in the absence or presence of interleukin (IL)-1beta or triptolide. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid-heat hydrolysis of the culture supernatants. The activities of matrix metalloproteinase (MMP)-1 and plasmin were measured with the specific substrates thiopeptolide and S-2251, respectively. The release of MMPs into the culture supernatant was assessed by immunoblot analysis and gelatin zymography, and the abundance of MMP mRNAs in the cells was determined by reverse transcription and real-time polymerase chain reaction., Results: Triptolide inhibited the IL-1beta-induced degradation of collagen by corneal fibroblasts in a dose- and time-dependent manner. Neither the activity of purified recombinant MMP-1 nor that of plasmin in culture supernatants was affected by triptolide. The IL-1beta-induced expression of MMP-1, -2, -3, and -9 by corneal fibroblasts was inhibited by triptolide at the protein or mRNA level., Conclusions: Triptolide inhibits collagen degradation by corneal fibroblasts by inducing downregulation of the production of MMPs, without directly affecting the collagenolytic activity of these enzymes.

Objective: To explore the effect of connective tissue growth factor on the pathogenesis of hypertrophic scar and keloid tissue., Methods: The content of hydroxyproline was determined and the expression of connective tissue growth factor gene was detected by the reverse transcription-polymerase chain reaction and image analysis technique in 5 normal skins, 15 hypertrophic scars and 7 keloid tissues., Results: The contents of hydroxyproline in the hypertrophic scar (84.10 +/- 1.76) and keloid tissue (92.38 +/- 2.04) were significantly higher than that of normal skin tissue (26.52 +/- 4.10) (P < 0.01). The index of connective tissue growth factor mRNA in the hypertrophic scar (0.78 +/- 0.63) and keloid tissue (0.84 +/- 0.04) were higher than that of normal skin tissue (0.09 +/- 0.25) (P < 0.01)., Conclusion: Connective tissue growth factor may play an important role in promoting the fibrotic process of hypertrophic scar and keloid tissue.

There is evidence that chronic exposure to high levels of ambient particulate pollutants (PM) is associated with chronic airflow obstruction, but how this occurs is not known. We exposed rat tracheal explants to Ottawa urban air particles (ECH93) or diesel exhaust particles. After 7 d in air organ culture, both types of PM increased explant procollagen and transforming growth factor (TGF)-beta 1 gene expression, and markedly increased tissue hydroxyproline. For both types of particle, nuclear factor-kappa B inhibitor SN50 completely blocked increased gene expression. With EHC93, procollagen expression was inhibited by the oxidant scavenger, tetramethylthiourea, and by the iron chelator, deferoxamine, but TGF-beta1 expression was not inhibited by deferoxamine. Inhibitors of extracellular signal regulated kinase and p38 kinase did not affect EHC93-induced gene expression. With diesel exhaust particles, tetramethylthiourea and deferoxamine had no effect, but extracellular signal regulated kinase and p38 inhibitors completely blocked effects on procollagen and TGF-beta 1. Fetuin, an inhibitor of TGF-beta receptor binding, prevented increases in procollagen gene expression. We conclude that two common types of PM can directly induce expression of genes involved in fibrogenesis and actual airway wall fibrosis through nuclear factor-kappa B- and TGF-beta-mediated mechanisms. PM-induced airway wall remodeling may play an important role in producing airflow obstruction in individuals living in high PM regions.

Background/aims: Oxidative stress is presumed to play an important role in hepatic fibrogenesis. Diets high in polyunsaturated fatty acids (PUFA) enhance fibrosis and have been associated with increased oxidative damage in some models of liver injury. The aim of this study was to determine the effects of dietary fat of varying PUFA content on iron-induced oxidative stress and fibrosis., Methods: Rats were given parenteral iron and diets supplemented with coconut oil, safflower oil or menhaden oil., Results: Hepatic iron overload was associated with induction of heme oxygenase-1, a sensitive indicator of oxidative stress, and with modest increases in hydroxyproline and procollagen I mRNA levels without histologically evident fibrosis, all of which were unaffected by dietary fat. In addition, iron loading was associated with increases in cysteine, gamma-glutamylcysteine and glutathione. Dietary fat brought about the expected alterations in peroxidizability, but did not alter indices of oxidative damage., Conclusion: These data highlight the distinction between oxidative stress and oxidative damage and suggest that the former is not sufficient to elicit overt fibrosis. Furthermore, while hepatic iron overload leads to oxidative stress, there is an associated upregulation of antioxidant defenses involving thiol metabolism that may be a critical factor limiting the accumulation of oxidative damage.

Objective: To investigate the effect of cartilage-derived growth factor (CDGF) on cultured rabbit chondrocytes, and the relation between CDGF, insulin-like growth factor-I (IGF-I), and proliferation and metabolism of chondrocytes., Methods: CDGF was extracted from chicken xiphoid in our laboratory. Chondrocytes were isolated from 3-week-old New Zealand male rabbits, and cultured in Ham's F-12 medium containing 10% fetal calf serum. The chondrocyts of second generation were treated with CDGF and/or IGF-I of different concentrations. With chloramine T method and MTT method, we compared the content of hydroxyproline in Ham's F-12 medium and observed proliferation and energy synthesis of chondrocytes., Results: CDGF and IGF-I both stimulated the proliferation and synthesis of hydroxyproline of chondrocytes dose-dependently. The optimal concentration of CDGF was 16 ng/ml and 32 ng/ml respectively, and that of IGF-I was 30 ng/ml. There was obvious synergic effect between CDGF and IGF-I., Conclusion: CDGF can stimulate the proliferation and collagen synthesis of chondrocytes and has synergistic effect with IGF-I.

Purpose: To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated., Methods: Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis., Results: The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts., Conclusion: These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.

Osteoblasts are the primary cells responsible for bone formation and are thought to originate from mesenchymal osteoprogenitor cells within skeletal tissues. To elucidate the osteoblastic differentiation process, fetal rat calvariae (FRC) were enzymatically digested and fractionated to provide an osteoprogenitor-enriched cell population. The third fraction of cells from the five sequential digestions tested showed a significant osteogenic response to dexamethasone (Dex), a well-known differentiation hormone, which was demonstrated by high alkaline phosphatase activity early in culture and enhanced calcium deposition and bone nodule formation in late stage cultures. These data indicate that fraction three contains a large number of osteoprogenitor cells. During the osteoblastic differentiation of the third fraction of FRC cells, the formation of collagen cross-links (pyridinoline and deoxypyridinoline) was time-dependently accelerated with the accumulation of collagens, which coincided with an onset of mineralization of the cultures, i.e., calcium deposition and bone nodule formation. Moreover, noncollagenous matrix proteins, bone sialoprotein and osteocalcin, were also increased at both mRNA and protein level in Dex-treated cultures with advancing culture periods. Further examination for mRNA expression of bone morphogenetic proteins (BMPs) and TGF-beta1 revealed a notable elevation in BMP-6 mRNA expression on days 3 and 10, and no significant change in TGF-beta1 expression. These observations suggested that the progressive formation of collagen cross-links, production of noncollagenous proteins, and up regulation of BMP-6 mRNA play an important role in the osteoblastic differentiation process of osteoprogenitor cells isolated from FRC. This culture system provides us a suitable model for in vitro bone formation.

There is increasing evidence for the presence and activity of methanotrophic bacteria in saline and alkaline aquatic environments located in different ecogeographical regions. Alkalitolerant halophilic and alkaliphilic halotolerant methanotrophs of type I were found to be able to utilize methane and methanol, to oxidize ammonium ions, and to transform various organic compounds in a wide range of water salinities (up to 12% NaCl) and pH values (from 5 to 11). The ecophysiological importance of methanotrophs in microbial communities inhabiting saline and alkaline aquatic environments is due to their involvement in the global cycles of methane and major bioelements (C, N, and S). Specific cyto- and biochemical properties of haloalkaliphilic methanotrophs--the synthesis of osmoprotectants (ectoine, 5-hydroxyproline, and sucrose), the accumulation of potassium ions, the formation of glycoprotein S layers on the outer surface of their cell walls, and the modification of the chemical composition of their membranes--allow them to adapt to highly saline and alkaline habitats. Due to their specific properties, haloalkaliphilic methanotrophs may become of use in modern biotechnology.

This paper describes the results of a comparative screening between the nematophagous Antarctic fungus Arthrobotrys tortor and other species of that genus for the production of extracellular collagenases. The nematode species used in this study was Caenorhabditis elegans, feeding on Escherichia coli cultures. Determination of collagenase activity was made using insoluble collagen from bovine Achilles tendon and determining the amount of solubilized hydroxyproline produced. The results show that the total amount of collagenase produced by the Antarctic strain of A. tortor was about threefold higher than that observed for the other species. In the Antarctic strain, collagenase was shown to be a constitutive enzyme.

Photopolymerizable polyethylene glycol (PEG) derivatives have been investigated as hydrogel tissue engineering scaffolds. These materials have been modified with bioactive peptides in order to create materials that mimic some of the properties of the natural extracellular matrix (ECM). The PEG derivatives with proteolytically degradable peptides in their backbone have been used to form hydrogels that are degraded by enzymes involved in cell migration, such as collagenase and elastase. Cell adhesive peptides, such as the peptide RGD, have been grafted into photopolymerized hydrogels to achieve biospecific cell adhesion. Cells seeded homogeneously in the hydrogels during photopolymerization remain viable, proliferate, and produce ECM proteins. Cells can also migrate through hydrogels that contain both proteolytically degradable and cell adhesive peptides. The biological activities of these materials can be tailored to meet the requirements of a given tissue engineering application by creating a mixture of various bioactive PEG derivatives prior to photopolymerization.

Aim: To clarify whether collagen deposition and collagen synthesis are increased in renovascular hypertensive rats (RHR) and whether these are affected by treatment with tetrandrine (Tet)., Methods: Collagen deposition was detected by electron microscope and evaluated by the amount of hydroxyproline in aorta of sham-operated rats (Sham), renovascular hypertensive rats (RHR), and Tet treated RHR. The aortic vascular smooth muscle cells (AVSMCs) were isolated and cultured. The collagen synthesis in cultured AVSMCs was detected by [3H]-proline incorporation., Results: The ultrastructure of aorta suggests that AVSMCs in RHR had been transferred from contractile phenotype to synthetic phenotype. Compared with sham-operated rats, the hydroxyproline content in aorta was increased by 36.6% in RHR, while it was reduced by 18.7% in Tet treated RHR (P < 0.01, vs RHR). Tet (0.1-10 mumol.L-1) treatment in vitro induced a concentration dependent depression [3H]-proline incorperation stimulated by NE or Ang II in AVSMCs from either Sham or RHR., Conclusion: The depressive effects of tetrandrine on collagen synthesis and deposition in arteries may contribute to the regression of vascular remodeling.

Addition of the amino acids threonine, serine, proline, and arginine to fermentations of the fungus Glarea lozoyensis influenced both the pneumocandin titer and the spectrum of analogues produced. Addition of threonine or serine altered the levels of the "serine analogues" of pneumocandins B(0) and B(5) and allowed for their isolation and identification. Proline supplementation resulted in a dose-dependent increase in the levels of pneumocandins B(0) and E(0), whereas pneumocandins C(0) and D(0) decreased as a function of proline level. Moreover, proline supplementation resulted in an overall increase in the synthesis of both trans-3- and trans-4-hydroxyproline while maintaining a low trans-4-hydroxyproline to trans-3-hydroxyproline ratio compared to the unsupplemented culture. Pneumocandin production and the synthesis of hydroxyprolines was also affected by addition of the proline-related amino acid arginine but not by the addition of glutamine or ornithine. Zinc, cobalt, copper, and nickel, trace elements that are known to inhibit alpha-ketoglutarate-dependent dioxygenases, affected the pneumocandin B(0) titer and altered the levels of pneumocandins B(1), B(2), B(5), B(6), and E(0), analogues that possess altered proline, ornithine, and tyrosine hydroxylation patterns.

Wound contraction and scar formation after cleft palate surgery impair maxillary growth and dentoalveolar development. Since myofibroblast numbers and scar formation are reduced by interferon-gamma (IFN-gamma) in the healing of dermal wounds, the hypothesis was tested that local administration of IFN-gamma reduces the numbers of myofibroblasts and the elevated amount of collagen during palatal mucoperiosteal wound healing. Standardized mucoperiosteal excision wounds were made in the palatal mucoperiosteum of young rats. Either IFN-gamma or vehicle alone (sham group) was repeatedly injected into the wound site between 4 and 29 days post-wounding. The results were compared with unmanipulated control wounds. Samples of wound tissue were prepared for biochemical and microscopic analysis. The hydroxyproline, sulfated glycosaminoglycan and DNA contents of the wound tissues were analyzed biochemically. The degree of re-epithelialization, tissue thickness, the numbers of myofibroblasts, and the amounts of elastin and collagen types I and III were evaluated on histological sections. Injection of vehicle alone affected almost all healing parameters, compared with the controls, and delayed the wound-healing process. IFN-gamma stimulated re-epithelialization and decreased the numbers of myofibroblasts when compared with vehicle-treated wounds. It also decreased the hydroxyproline and glycosaminoglycan contents of 60-day-old wound tissue, but the histological characteristics of scar tissue persisted. Therefore, IFN-gamma is able to reduce the numbers of myofibroblasts and the collagen content of scar tissue after palatal wound healing. It may be a promising pharmaceutical agent for the reduction of wound contraction and scarring after cleft palate surgery.