Insight into the spoilage heterogeneity of meat-borne bacteria isolates with high-producing collagenase

Chilled chicken is inevitably contaminated by microorganisms during slaughtering and processing,resulting in spoilage.Cutting parts of chilled chicken,especially wings,feet,and other skin-on products,are abundant in collagen,which may be the primary target for degradation by spoilage microorganisms.In this work,a total of 17 isolates of spoilage bacteria that could secrete both collagenase and lipase were determined by raw-chicken juice agar(RJA)method,and the results showed that 7 strains of Serratia,Aeromonas,and Pseudomonas could significantly decompose the collagen ingredients.The gelatin zymography showed that Serratia liquefaciens(F5)and(G7)had apparent degradation bands around 50 kDa,and Aeromonas veronii(G8)and Aeromonas salmonicida(H8)had a band around.65 and 95 kDa,respectively.The lipase and collagenase activities were detected isolate-by-isolate,with F5 showing the highest collagenase activity.For spoilage ability on meat in situ,F5 performed strongest in spoilage ability,indicated by the total viable counts,total volatile basic nitrogen content,sensory scores,lipase,and collagenase activity.This study provides a theoretical basis for spoilage heterogeneity of strains with high-producing collagenase in meat.

Long-term assessment of collagenase treatment for Dupuytren’s contracture:A 10-year follow-up study

BACKGROUND Enzymatic fasciotomy with collagenase clostridium histolyticum(CCH)has revolutionized the treatment for Dupuytren’s contracture(DC).Despite its benefits,the long-term outcomes remain unclear.This study presented a comprehensive 10-year follow-up assessment of the enduring effects of CCH on patients with DC.AIM To compare the short-term(12 wk)and long-term(10 years)outcomes on CCH treatment in patients with DC.METHODS A cohort of 45 patients was treated with CCH at the metacarpophalangeal(MCP)joint and the proximal interphalangeal(PIP)joint and underwent systematic reevaluation.The study adhered to multicenter trial protocols,and assessments were conducted at 12 wk,7 years,and 10 years post-surgery.RESULTS Thirty-seven patients completed the 10-year follow-up.At 10 years,patients treated at the PIP joint exhibited a 100%recurrence.However,patients treated at the MCP joint only showed a 50%recurrence.Patient satisfaction varied,with a lower satisfaction reported in PIP joint cases.Recurrence exceeding 20 degrees on the total passive extension deficit was observed,indicating a challenge for sustained efficacy.Significant differences were noted between outcomes at the 7-year and 10-year intervals.CONCLUSION CCH demonstrated sustained efficacy when applied to the MCP joint.However,caution is warranted for CCH treatment at the PIP joint due to a high level of recurrence and low patient satisfaction.Re-intervention is needed within a decade of treatment.

A randomized clinical trial assessing the efficacy of single and multiple intralesional collagenase injections for treating contracted scars

Background:Scar contractions caused by trauma or burns can cause secondary physical dysfunction and disfigurement.Many minimally invasive methods for scar contraction have shown limited applicability and efficacy.This study investigated the feasibility and efficacy of intralesional collagenase injections for scar contraction treatment.Methods:Patients with contracted scars who had limited joint movement and physical disfiguration for>1 year were enrolled in this single-blind,randomized clinical trial from July 2017 to February 2018 at Shanghai Ninth People’s Hospital.Collagenase was injected into the firm-contracted scar(15 U/cm^(2))three times at 4-week intervals in the multiple treatment group and once in the single treatment group,and a placebo injection was performed in the control group.Scar length and skin texture were documented at the 4-and 12-week follow-ups.The safety of the collagenase treatment was also evaluated.Results:The contracted scar was significantly elongated after both single and multiple collagenase treatments.The results showed that,compared to a one-time treatment,repeated injections were more effective at 12 weeks,with an average improvement of 26.83(15.79%).At 12 weeks,78.9% of the patients in the multiple group and 52.9%in the single group achieved significant improvement at 12 weeks.No severe adverse events were observed.Conclusion:Intralesional collagenase injection showed promising results in improving scar contraction and provides an alternative treatment for patients.

A novel arterial pouch model of saccular aneurysm by concomitant elastase and collagenase digestion

Background: An ideal aneurysm model of cerebral aneurysm is of great importance for studying the pathogenesis of the lesion and testing new techniques for diagnosis and treatment. Several models have been created in rabbits and are now widely used in experimental studies; however, every model has certain intrinsic limitations. Here we report the development of a novel saccular aneurysm model in rabbits using an arterial pouch that is subject to in vitro pre-digestion with combined elastase and collagenase. Methods: A segment of right common carotid artery (CCA) was dissected out and treated with elastase (60 U/ml, 20 min) followed by type I collagenase (1 mg/ml, 15 min) in vitro. The graft was anastomosed to an arterial arch built with the left CCA and the remaining right CCA, while the other end of the graft was ligated. The dimension and tissue structure of the pouch were analysed immediately, 2 or 8 weeks after operation. Findings: Ten terminal aneurysms were produced. The gross mor-phology of the aneurysm resembles the human cerebral terminal aneurysms. We have observed the following pathological changes: (1) growth of the aneurysm (mean diameter increased from (2.0±0.1) to (3.2±0.3) mm at 2 weeks, P【0.001, n=7~10); (2) thinning of the aneurysmal wall (the mean wall thickness decreased to 44% at 2 weeks), which was accompanied by significant losses of elastic fibres, collagen and the cellular component; and (3) spontaneous rupture (3 out of 9, one aneurysm ruptured 24 h after operation with the other two at 2 and 4 weeks respectively). Conclusion: This rabbit arterial pouch model mimics human cerebral aneurysms in relation to morphology and histology. In particular, this model exhibited an increased tendency of spontaneous rupture.

Impact of vasculature damage on the outcome of spinal cord injury:a novel collagenase-induced model may give new insights into the mechanisms involved

The deleterious effect of vasculature damage on the outcome of spinal cord injury has long been recognized, and numerous clinical studies have shown that the presence of hemorrhage into the spinal cord is directly associated with a poorer neurological outcome. Vascular damage leads to de- creased blood flow to the cord and the release of potentially toxic blood-borne components. Here we consider the mechanisms that may be contributing to hemorrhage-induced damage and discuss the utility of a new model of spinal cord hemorrhage, which was urgently required as most of our current understanding has been extrapolated from intracerebral hemorrhage studies.

Isolation and activation of collagenase from fish processing waste

Collagenase was isolated from fish waste (a mixture of haddock, herring, ground fish and flounder) using a Tris-buffer system. The proteins in the crude extract were precipitated using ammonium sulfate (40% - 80%) and purified with gel-filtration chromatography using Sephadex G-100. The results showed that the collagenase enzyme was produced as a latent enzyme and was activated with bovine trypsin and potassium thiocyanate (KSCN). The enzyme activity was affected by pH and temperature. Optimal enzyme activities were found at 35?C and a pH of 7.5 when insoluble collagene type I was used as substrate and the liberated amino acids were measured in relation to L-leucine in the presence of ninhydrin. The enzyme activity was completely inhibited by the action of ethylenediaminetetraacetic acid (EDTA) suggesting that the collagenase enzyme isolated from the fish waste is a metalloproteinase enzyme requiring metal ions for enzyme activity. Dialysis against KSCN decreased the enzyme total activity and increased its specific activity. Sodium dodecyl sulphate polyacryla-mide gel electrophoresis (SDS-PAGE) indicated that the purified procollagenase enzyme have only one band at molecular weight of 50 kilodaltons (kDa). When the enzyme was cleaved with trypsin, it was found to consist of two subunits: a large unit with a molecular weight of 50 kDa and a small unit with a molecular weight of 10 kDa.

The Role of Neutrophil Collagenase in Endotoxic Acute Lung Injury

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L 1), 6 h (group L 2), 12 h (group L 3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L 1, L 2, L 3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L 1, L 2, L 3) were also significantly higher than that of control group (P<0.01). Moreover, among group L 1, L 2 and L 3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.

Therapeutic applications of collagenase(metalloproteases): A review

Non-invasive therapeutic methods have recently been used in medical sciences. Enzymes have shown high activity at very low concentrations in laboratories and pharmaceutical,enabling them to play crucial roles in different biological phenomena related to living organism, especially human medicine. Recently, using the therapeutic methods based on non-invasive approaches has been emphasized in medical society. Researchers have focused on producing medicines and tools reducing invasive procedures in medical.Collagenases are proteins which catalyze chemical processes and break the peptide bonds in collagen. Collagen may be generated more than the required amount or produced in unsuitable sites or may not degrade after a certain time. In such cases, using an injectable collagenase or its ointment can be helpful in collagen degradation. In both in vitro and in vivo tests, it has been revealed that collagenases have several therapeutic properties in wound healing, burns, nipple pain and some diseases including intervertebral disc herniation, keloid, cellulite, lipoma among others. This review describes the therapeutic application of collagenase in medical sciences and the process for its production using novel methods, paving the way for more effective and safe applications of collagenases.

Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates

A selective, sensitive, and convenient assay for human collagenase is required because of its implication in diseases such as rheumatoid arthritis, osteoarthritis, and tumors. Here, a novel assay for human collagenase activity is described in which enzymatic degradation of native collagen or acetyl peptide is determined by using a fluorogenic reaction for oligopeptides. The oligopeptides are quantified spectrofluorometrically with either 3,4-dihydroxyphenylacetic acid or 1,2-dihydroxybenzen reaction in the presence of sodium periodate and sodium borate (pH 7 - 8). These reactions can selectively convert N-terminal Gly-containing oligopeptides and N-terminal Ile-containing oligopeptides to fluorescence (FL) compounds, respectively, but not proteins, acetyl peptides or amino acids. Under optimized conditions using 1.65 μM collagen IV or 1.5 mM Ac-GPQGI- AGQ as substrates, this assay exhibits a proportional relationship between FL intensities and human collagenase-3 (MMP-13) concentrations. It can assay endogenous collagenase activities in several biological samples, such as cultured human cells and cheek tissue.

Use of intralesional collagenase in the treatment of peyronie's disease: A review

AIM:To review the relevant literature in an effort to examine the body of evidence available to date.METHODS:Ovid MEDLINE search database was queried using MeS H terms"penile induration","peyronie’s disease","Collagenases"and"Collagenase"using various permutations.No temporal parameters were employed.RESULTS:In all,5 relevant clinical trials were isolated from 34 results.These trials were analyzed using the Oxford Centre for Evidence-Based Medicine criteria.They were further examined based on study design and methods;the primary and secondary outcomes were reviewed for treatment efficacy and collagenase-related side effects.CONCLUSION:Intralesional collagenase appears to be safe and effective in the non-surgical treatment of Peyronie’s disease.However,the data remains limited and further inquiries into the safety of collagenase,treatment standardization and standardized outcomesreporting remain necessary.Furthermore,studies comparing intralesional collagenase to alternative medical and surgical therapy will be important in guiding the future treatment decision process.

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by Clostridium histolyticum

Clostridium histolyticum is used for production of several proteolytic enzymes such as elastase, neutral proteases, clostripain and in particular collagenase. Besides industrial applications, collagenase has been indispensable for medical purposes including isolation of pancreatic islets for diabetes treatment. The aim of this study was to optimize the method for production and partial purification of a new collagenase blend and to test its suitability for successful pancreatic islets isolation in a rat model. Bacterial strain of C. histolyticum was sequenced for presence of the collagenase genes. Different fermentation conditions were tested and the process of collagenase extraction was modified and optimized. Samples of collagenases were taken for western blot detection, activity assessment, and ability for dissociation of pancreatic tissue. Findings indicate that concentrated trypton growth medium with pepton was the most suitable for Clostridium growth and collagenase production. Whole genome sequencing revealed two genes for collagenase and also gene for clostripain. Western blot specific detection helped to select useful production modifications. Following these modifications was also improved the yield, morphology and in vitro function of intact pancreatic islets which were finally comparable or better than those achieved using standard blends of collagenase. The results support the use of the new collagenase blend for islet isolation giving thus the opportunity to choose an alternative product. Our next steps would lead to further enzyme purification through scaling up of the production method for a wider use.

Antitumor effects of the molecule-downsized immunocon-jugate composed of lidamycin and Fab' fragment of monoclonal antibody directed against type IV collagenase

Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab′-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab′ fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab′-LDM conjugate was 65 kD with a 1:1 molecular ratio of Fab′ and LDM. The Fab′-LDM conjugate maintained most part of the immunoreactivity of Fab′ fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab′-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab′-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment I, the drugs were given intravenously on day 1 and day 8. Fab′-LDM at the doses of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg inhibited tumor growth by 76.7%, 93.3% and 94.8%, while free LDM at 0.05 mg/kg inhibited tumor growth by 76.1%, respectively. In experiment II, the drugs were given intravenously on day 4 and day 11, Fab′-LDM at the doses of 0.025 mg/kg and 0.05 mg/kg inhibited tumor growth by 74.2%, 80.9%, while free LDM at 0.05 mg/kg inhibited tumor growth by 60.5%, respectively. In terms of survival time, Fab′-LDM was more effective than free LDM. The results suggest that the molecule-downsized immunoconjugate directed against type IV collagenase is of high efficacy in experimental cancer therapy.

Production and characterization of a human single-chain Fv to collagenase IV

The over\|expression of collagenase IV in tumor tissues was found to be closely related to tumor metastasis. Collagenase IV has been therefore considered as one of the novel indicative molecules for tumor diagnosis and treatment. Based on phage display antibody library technique, a single\|chain Fv specific for collagenase IV was successfully cloned. This antibody, referred to as hCo4, was mainly composed of variable regions from heavy and light chains, with its molecular weight of 27 ku. The engineered antibody bound to collagenase IV specifically. The affinity of hCo4 was found to be the same as that of a single\|chain antibody constructed from a monoclonal antibody to collagenase IV. Since hCo4 is the smallest among all the antibodies specific for collagenase IV and it is of human origin, it has a potential to be applied for tumor immunotherapy and for the study of the relationship between collagenase IV and tumor metastasis.

Intracellular expression of a single-chain antibody directed against type IV collagenase inhibits the growth of lung cancer xenografts in nude mice

It was documented that type IV collagenase with two subtypes of 72 ku/MMP-2 and 92 ku/MMP-9 plays an important role in tumor invasion and metastasis. The endoplasmic reticulum (ER)- retained, single chain Fv antibody fragment (scFv) was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acids (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector. The resulting plasmid was sequenced and then introduced into PG cells, a highly metastatic human lung cancer cell line, by lipofectAMINE method. The result of intrabody gene therapy showed that type IV collegenase expression was down regulated significantly as measured by ELISA. The biological behavior of PG cell, such as the ability of in vitro invasion through Matrigel, colony formation on soft agar, was also inhibited by scFv M97 transfection. Animal experiments in a xenograft model of human lung

溶藻弧菌实时荧光定量PCR快速检测方法的建立

为建立溶藻弧菌(VA)的快速检测方法,本研究以VA Collagenase基因为靶基因设计合成引物及Taq Man探针,建立了实时荧光定量PCR快速检测VA的方法。结果显示,对15株实验菌株进行荧光定量PCR检测,只有VA菌株检测为阳性,表明该检测方法特异性强;该方法的灵敏度为18 cfu/m L;稳定性和重复性实验结果表明,同一样品重复检测4次Ct值的变异系数均小于2%;利用该检测方法对采集的165份样品进行检测,共计检出2份VA阳性样品,与SN/T2564-2010行标法检测结果一致,显示了良好的实用性。该检测方法灵敏度高、特异性强,具有良好的实用性。

具胶原蛋白酶活性铜绿假单胞菌的筛选

从成都皮革厂等堆积废弃毛皮、皮革的场所采集土样 ,通过以明胶为主要基质培养基进行富集和初筛 ,获得 95株有明胶酶活性菌株。挑选其中 2 8株明胶酶活性较高的菌株进行牛皮消化试验 ,有 1 2株菌能在 48h内完全消化小牛皮。以III型酸溶性胶原为底物 ,测定了 1 2株菌发酵培养液中胶原蛋白酶活性 ,确认这 1 2株菌都具有胶原蛋白酶活性 ,酶活力基本相同 ,约 1 0～ 1 6U mL。经形态观察、生理生化特征分析及BIOLOG微生物鉴定仪鉴定 ,这 1 2株菌分为两类 ,分别是铜绿假单胞菌 (Pseudomonasaeraginosa)和火神发光杆菌 (Photobacteriumlogei) (结果另文发表 )。对铜绿假单胞菌产生胶原蛋白酶粗酶性质进行了研究 ,其酶活最适温度为 32℃ ,最适pH为 7 5 ,可以被金属蛋白酶抑制剂EDTA和EGTA部分抑制 ,不能被PMSF抑制。对铜绿假单胞菌产胶原蛋白酶发酵条件的研究表明 ,不仅培养基中氮源、碳源和金属离子影响产酶量 ,而且发酵工艺对胶原蛋白酶的产生也有较大影响。

Effects of glycyrrhetinic acid on collagen metabolism of hepatic stellate cells at different stages of liver fibrosis in rats

INTRODUCTIONLiver fibrosis is a dynamic course leading tocirrhosis from a various chronic liver diseases. Thepathological basis of fibrosis is the disturbance ofproduction and degradation of the extracellularmatrix (ECM), which causes accumulation of ECMin the liver[1,2].

Modified insulin-like growth factor 1 containing collagen-binding domain for nerve regeneration

Insulin-like growth factor 1 （IGF-I） is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve targeted and long-lasting treatment, we investigated the addition of a binding structure by fusing a collagen-binding domain to IGF- 1. After confirming its affinity for collagen, the biological activity of this construct was examined by measuring cell proliferation after transfection into PC12 and Schwann cells using a 3-（4,5-dimethyl-2-thiazolyl）-2,5-di- phenyl-2-H-tetrazolium bromide assay. Immunofluorescence staining was conducted to detect neurofilament and microtubule-associated protein 2 expression, while real time-polymerase chain reaction was utilized to determine IGF-1 receptor and nerve growth/actor mRNA expression. Our results demonstrate a significant increase in collagen-binding activity of the recombinant protein compared with IGF-1. Moreover, the recombinant protein promoted proliferation of PC12 and Schwann cells, and increased the expression of neurofilament and microtubule-associated protein 2. Importantly, the recombinant protein also stimulated sustained expression of IGF-1 receptor and nerve growth factor mRNA for days. These results show that the recombinant protein achieved the goal of targeting and long-lasting treatment, and thus could become a clinically used factor for promoting nerve regeneration with a prolonged therapeutic effect.

Analysis of isolation of cerebral cortical neurons in rats by different methods

The aim of this study was to find a way to efficiently separate neuronal cells from the cerebral cortex of adult rats,providing a reference method for rapid acquisition of neuronal cells from the adult rat brain.Fifteen SD rats were randomly divided into three groups,with five SD rats in each group.Then,neuron cells were isolated from the adult rat cerebral cortex by the grinding method,the trypsin method,and the collagenase II method,respectively.The expression of anti-NeuN in the neurons of each group was analyzed by flow cytometry.The acquisition rates and morphology of neurons of each group were observed by immunofluorescence staining.The grinding or collagenase II method is more suitable for rapid acquisition of neuronal cells from an adult rat’s cerebral cortex.The number of neuron cells obtained by the trypsin method were very few,so it is not convenient for later experiments.

Wound healing activity of Ullucus tuberosus,an Andean tuber crop

Objective:This study was designed to investigate the wound healing activity of aqueous extracts of Ullucus tuberosus(U.tuberosus) using in vitro models.Methods:Lyophilized pulp and acetone extracts of U.tuberosus were produced using ultrasound extraction.The capacity for collagenase activation was evaluated using fluorescence detection of the enzymatic activity.Then,the influence of U.tuberosus extracts on cell proliferation,cell migration and synthesis of the extracellular matrix(ECM)proteins,metalloproteinase(MMP-1) and pro-collagen was analyzed using human dermal fibroblasts in culture.Results:An increase in collagenase activity of 12% supports the utility of U.tuberosus as an agent for scar treatment.In addition,the extracts showed an increase in the proliferation and migration of human dermal fibroblasts and the production of pro-collagen and MMP-1 after treatment with U.tuberosus extracts.The increase in proliferation,migration and pro-collagen levels positively influenced the regeneration of scarless tissue during the proliferation phase,whereas the increase in MMP-1 may have favored the wound healing process during the remodeling and cellular differentiation phases.Conclusion:The results of this study show for first time that U.tuberosus is a promising candidate to support scarless tissue regeneration.