Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Bacterial chitinases: genetics, engineering and applications.

Chitinases are a group of enzymes that catalyze chitin hydrolysis and are present in all domains of life. Chitinases belong to different glycosyl hydrolase families with great diversity in their sequences. Microorganisms such as bacteria and fungi produce chitinases for nutrition, and energy, and to parasitize the chitinous hosts. But chitinases from bacteria are of special interest due to their ubiquitous nature and ability to perform under extreme conditions. Chitinases produced by bacteria have been explored for their use in agriculture and industry. In agriculture, their main role is to control chitin-containing insect pests, fungal pathogens, and nematodes. In the seafood industry, they found their role in the management of processing wastes which are mainly chitinous substances. Chitinases are also used to synthesize low molecular weight chitooligomers which are proven bioactive compounds with activities such as anti-tumour, antimicrobial, and immunity modulation. Considering their importance in ecology and biotechnological applications, several bacterial chitinases have been studied in the last two decades. Despite their potential, bacterial chitinases have a few limitations such as low production and lack of secretion systems which make the wild-type enzymes unfit for their applications in industries and other allied sectors. This review is an attempt to collate significant works in bacterial chitinases and their application in various industries and the employment of various tools and techniques for improvement to meet industrial requirements.

Functional expression of recombinant hybrid enzymes composed of bacterial and insect's chitinase domains in E. coli.

To elucidate the functional alteration of the recombinant hybrid chitinases composed of bacterial and insect's domains, we cloned the constitutional domains from chitinase-encoding cDNAs of a bacterial species, Bacillus thuringiensis (BtChi) and a lepidopteran insect species, Mamestra brassicae (MbChi), respectively, swapped one's leading signal peptide (LSP) - catalytic domain (CD) - linker region (LR) (LCL) with the other's chitin binding domain (ChBD) between the two species, and confirmed and analyzed the functional expression of the recombinant hybrid chitinases and their chitinolytic activities in the transformed E. coli strains. Each of the two recombinant cDNAs, MbChi's LCL connected with BtChi's ChBD (MbLCL-BtChBD) and BtChi's LCL connected with MbChi's ChBD (BtLCL-MbChBD), was successfully introduced and expressed in E. coli BL21 strain. Although both of the two hybrid enzymes were found to be expressed by SDS-PAGE and Western blotting, the effects of the introduced genes on the chitin metabolism appear to be dramatically different between the two transformed E. coli strains. BtLCL-MbChBD remarkably increased not only the cell proliferation rate, extracellular and cellular chitinolytic activity, but also cellular glucosamine and N-acetylglucosamine levels, while MbLCL-BtChBD showed about the same profiles in the three tested subjects as those of the strains transformed with each of the two native chitinases, indicating that a combination of the bacterial CD of TIM barrel structure with characteristic six cysteine residues and insect ChBD2 including a conserved six cysteine-rich region (6C) enhances the attachment of the enzyme molecule to chitin compound by MbChBD, and so increases the catalytic efficiency of bacterial CD.

Anticancer and antifungal efficiencies of purified chitinase produced from Trichoderma viride under submerged fermentation.

Trichoderma viride AUMC 13021 isolated from Mangrove soil of Ras Mohammed protected area at Sharm El-Sheikh, Egypt, was optimized to promote chitinase activity under submerged fermentation. The maximum enzyme yield (38.33 U/mg protein) was obtained at 1.4% of colloidal chitin, 96 h of incubation, 35°C, pH 6.5 and 125, rpm and using maltose (1%) and yeast extract (1%) as supplementation of salt basal medium. The enzyme has been purified with an overall yield of 73.1% and 5.48 purification fold, and a specific activity of 210.16 U/mg protein. The molecular mass of the purified chitinase was 62 kDa. Maximal activity of chitinase was recorded at pH 6.5 and 40°C. The highest activity was recorded in the case of colloidal chitin, with an apparent Km value of 6.66 mg/ml and Vmax of 90.8 U/ml. The purified chitinase was activated by Ca2+ and Mn2+ while the activity was inhibited by Hg2+, Zn2+, Cu2+, Co2+, dodecyl sulphate and EDTA. In vivo, the median lethal dose (LD50) was approximately 18.43 mg/kg body weight of Sprague Dawley rats. MTT assay showed that the purified chitinase has a toxic effect to MCF7 with an IC50 value 20 µg/ml, and HCT-116 cell lines with an IC50 value 44 µg/ml. Moreover, the purified enzyme showed significant antifungal activity against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt.

Microbial chitinases: properties, current state and biotechnological applications.

Chitinases are a group of hydrolytic enzymes that catalyze chitin, nd are synthesized by a wide variety of organisms. In nature, microbial chitinases are primarily responsible for chitin decomposition. Several chitinases have been reported and characterized, and they are garnering increasing attention for their uses in a wide range of applications. In the food industry, the direct fermentation of seafood, such as crab and shrimp shells, using chitinolytic microorganisms has contributed to increased nutritional benefits through the enhancement of chitin degradation into chitooligosaccharides. These compounds have been demonstrated to improve human health through their antitumor, antimicrobial, immunomodulatory, antioxidant, and anti-inflammatory properties. Moreover, chitinase and chitinous materials are used in the food industry for other purposes, such as the production of single-cell proteins, chitooligosaccharides, N-acetyl D-glucosamines, biocontrol, functional foods, and various medicines. The functional properties and hydrolyzed products of chitinase, however, depend upon its source and physicochemical characteristics. The present review strives to clarify these perspectives and critically discusses the advances and limitations of microbial chitinase in the further production of functional foods.

Expression of M2 macrophage markers YKL-39 and CCL18 in breast cancer is associated with the effect of neoadjuvant chemotherapy.

PURPOSE: High activity of enzyme TOP2a in tumor cells is known to be associated with sensitivity to anthracycline chemotherapy, but 20% of such patients do not show clinical response. Tumor microenvironment, including tumor-associated macrophages (TAM), is an essential factor defining the efficiency of chemotherapy. In the present study, we analyzed the expression of M2 macrophage markers, YKL-39 and CCL18, in tumors of breast cancer patients received anthracycline-based NAC. METHODS: Patients were divided into two groups according to the level of doxorubicin sensitivity marker TOP2a: DOX-Sense and DOX-Res groups. Expression levels of TOR2a, CD68, YKL-39 and CCL18 genes were analyzed by qPCR, the amplification of TOR2a gene locus was assessed by the microarray assay. Clinical and pathological responses to neoadjuvant chemotherapy were assessed. RESULTS: We found that the average level of TOP2a expression in patients of DOX-Sense group was almost 10 times higher than in patients of DOX-Res group, and the expression of CD68 was 3 times higher in the DOX-Sense group compared to DOX-Res group. We demonstrated that expression levels of M2-derived cytokines but not the amount of TAM is indicative for clinical and pathological chemotherapy efficacy in breast cancer patients. Out of 8 patients from DOX-Sense group who did not respond to neoadjuvant chemotherapy (NAC), 7 patients had M2+ macrophage phenotype (YKL-39+CCL18- or YKL-39-CCL18+) and only one patient had M2- macrophage phenotype (YKL-39-CCL18-). In DOX-Res group, out of 14 patients who clinically responded to NAC 9 patients had M2- phenotype and only 5 patients had M2+ macrophage phenotype. Among pathological non-responders in DOX-Sense group, 19 (82%) patients had M2+ tumor phenotype and only 4 (18%) patients had M2- phenotype. In DOX-Res group, all 5 patients who pathologically responded to NAC had M2 phenotype (YKL-39-CCL18-). Unlike the clinical response to NAC, the differences in the frequency of M2+ and M2- phenotypes between pathologically responding and non-responding patients within DOX-Sense and DOX-Res groups were statistically significant. CONCLUSIONS: Thus, we showed that in patients with breast cancer who received anthracycline-containing NAC the absence of clinical response is associated with the presence of M2+ macrophage phenotype (YKL-39-CCL18 + or YKL-39 + CCL18-) based on TOP2a overexpression data.

Cadmium ion inhibition of quorum signalling in Chromobacterium violaceum.

Single-celled bacteria are capable of acting as a community by sensing and responding to population density via quorum signalling. Quorum signalling in Chromobacterium violaceum, mediated by the luxI/R homologue, cviI/R, regulates a variety of phenotypes including violacein pigmentation, virulence and biofilm formation. A number of biological and organic molecules have been described as quorum signalling inhibitors but, to date, metal-based inhibitors have not been widely tested. In this study, we show that quorum sensing is inhibited in C. violaceum in the presence of sub-lethal concentrations of cadmium salts. Notable Cd2+-inhibition was seen against pigmentation, motility, chitinase production and biofilm formation. Cd-inhibition of quorum-signalling genes occurred at the level of transcription. There was no direct inhibition of chitinase activity by Cd2+ at the concentrations tested. Addition of the cognate quorum signals, N-hexanoyl homoserine lactone or N-decanoyl homoserine lactone, even at concentrations in excess of physiological levels, did not reverse the inhibition, suggesting that Cd-inhibition of quorum signaling is irreversible. This study represents the first description of heavy metal-based quorum inhibition in C. violaceum.

Purification and biochemical characterization of novel acidic chitinase from Paenicibacillus barengoltzii.

The novel chitinase (PbChi67) from the marine bacterium Paenicibacillus barengoltzii CAU904 was purified and biochemically characterized. PbChi67 was purified to apparent homogeneity with 10.2 fold purification and 8.0% recovery yield. The molecular mass of the enzyme was 67.0kDa by SDS-PAGE and 67.9kDa by gel filtration, respectively. PbChi67 was most active at pH 3.5 and was stable within pH 3.0-9.0. The optimal temperature of PbChi67 was 60°C and it was stable up to 55°C with a thermal denaturing half-life of 43min at 65°C. The enzyme exhibited strict substrate specificity towards colloidal chitin and glycol chitin but showed no or trace activities towards other tested substrates. The Km and Vmax values of PbChi67 for colloidal chitin and glycol chitin were 3.35mg/mL and 17.1µmol/min/mg, and 2.66mg/mL and 15.0µmol/min/mg, respectively. PbChi67 hydrolyzed colloidal chitin to yield N-acetyl chitooligosaccharides (COSs) with degree of polymerization (DP) of 2-4 at the initial hydrolysis stage, indicating that it is an endo-type chitinase. These properties make the enzyme as a good candidate for recycling of chitin materials.

Effective and complex stimulation of the biodegradation system of fungus Cerrena unicolor by rapeseed meal fermentation.

The effect of supplementation of medium with rapeseed meal (RM) on production of biotechnologically important enzymes was investigated in submerged cultures of the white rot fungus Cerrena unicolor. The addition of RM (3.5% w/v) distinctly stimulated the activities of laccase, chitinase, and ß-glucosidase. As compared to the control, the activities of chitinase, ß-glucosidase, and laccase in the RM supplemented cultures were up to 4.1, 8.4, and 3.9 times higher, respectively. The results of the spectrophotometric and spectrofluorometric measurements were additionally confirmed by zymographic analysis of the samples. The level of sugars and phenolic compounds as well as the antioxidative ability of fungal preparations were also determined. The results obtained indicate that the submerged liquid fermentation of rapeseed meal can be proposed as an inexpensive and very effective method for biotechnological production of chitinase, ß-glucosidase, and laccase by C. unicolor.

Are cyclic lipopeptides produced by Bacillus amyloliquefaciens S13-3 responsible for the plant defence response in strawberry against Colletotrichum gloeosporioides?

UNLABELLED: The antagonistic strain Bacillus amyloliquefaciens strain S13-3 decreased the severity of strawberry anthracnose caused by Colletotrichum gloeosporioides. The foliar application of S13-3 triggered the expression of pathogenesis-related proteins, chitinase and ß-1,3-glucanase, in strawberry leaves. We identified lipopeptide antibiotics, including iturin A, fengycin, mixirin, pumilacidin and surfactin, produced and secreted by S13-3. Iturin A and surfactin elicited the gene expression of the pathogenesis-related proteins in strawberry leaves, suggesting that antagonistic strain S13-3 confers resistance to strawberry leaves through the production of lipopeptide antibiotics. In fact, iturin A and surfactin triggered induced systemic resistance on strawberry plants, resulting in the reduction of the severity of anthracnose disease caused by Colletotrichum gloeosporioides. The bifunctional activity of S13-3, which consists of the antagonistic effect and the induction of plant defence response by the antibiotics produced by it, may make S13-3 an innovative biological control agent against phytopathogens in strawberry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study tries to determine whether biocontrol of phytopathogens by Bacillus amyloliquefaciens in strawberry can be connected to induced plant resistance. The results suggested that the antagonistic strain B. amyloliquefaciens S13-3 confers resistance to strawberry through the production of lipopeptide antibiotics.

Transformation of blackgram (Vigna mungo (L.) Hepper) by barley chitinase and ribosome-inactivating protein genes towards improving resistance to Corynespora leaf spot fungal disease.

Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25-40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27-47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram.

Improved expression of single-chain antibodies in Ustilago maydis.

To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.

Acceleration of bone repair in NOD/SCID mice by human monoosteophils, novel LL-37-activated monocytes.

BACKGROUND: An incomplete understanding of bone forming cells during wound healing and ectopic calcification has led to a search for circulating cells that may fulfill this function. Previously, we showed that monoosteophils, a novel lineage of calcifying/bone-forming cells generated by treatment of monocytes with the natural peptide LL-37, are candidates. In this study, we have analyzed their gene expression profile and bone repair function. METHODS AND FINDINGS: Human monoosteophils can be distinguished from monocytes, macrophages and osteoclasts by their unique up-regulation of integrin α3 and down-regulation of CD14 and CD16. Monoosteophils express high mRNA and protein levels of SPP1 (osteopontin), GPNMB (osteoactivin), CHI3L1 (cartilage glycoprotein-39), CHIT1 (Chitinase 1), MMP-7, CCL22 and MAPK13 (p38MAPKδ). Monocytes from wild type, but not MAPK13 KO mice are also capable of monoosteophil differentiation, suggesting that MAPK13 regulates this process. When human monoosteophils were implanted in a freshly drilled hole in mid-diaphyseal femurs of NOD/SCID mice, significant bone repair required only 14 days compared to at least 24 days in control treated injuries. CONCLUSION: Human derived monoosteophils, characterized as CD45(+)α3(+)α3ß(+)CD34(-)CD14(-)BAP (bone alkaline phosphatase)(-) cells, can function in an animal model of bone injury.

Evaluation of AMCase and CHIT-1 expression in monocyte macrophages lineage.

Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT-1) are two active chitinases expressed in humans. The chitinase activity of AMCase was found to be causative in allergic inflammation and its expression was found to be induced by interleukin-13. CHIT1-1 is expressed by phagocytic cells and extremely high levels are seen in lysosomal storage diseases. Despite that AMCase expression in the inflammation is under investigation, little is known regarding its regulation during macrophages' full maturation and polarization. In this study, we compared AMCase and CHIT-1 modulation during monocyte to macrophage transition and polarization. Gene expression analysis was investigated by real-time PCR from mRNA of human monocytes obtained from buffy coat of healthy volunteers, from mRNA of polarized to classically activated macrophages (or M1), obtained by interferon (IFN)-γ and lipopolysaccharide (LPS) treatment, and from mRNA of alternatively activated macrophages (or M2) obtained by interleukin (IL)-4 exposure. Our results showed that the expression of AMCase and CHIT-1 were differently modulated in HMMs at different stage of maturation. The behavior of these two active chitinase suggests that in the immune response their role is complementary.

Fisetin, a bioactive flavonol, attenuates allergic airway inflammation through negative regulation of NF-κB.

Persistent activation of nuclear factor-κB (NF-κB) has been associated with the development of asthma. Fisetin (3,7,3',4'-tetrahydroxyflavone), a naturally occurring bioactive flavonol, has been shown to inhibit NF-κB activity. We hypothesized that fisetin may attenuate allergic asthma via negative regulation of the NF-κB activity. Female BALB/c mice sensitized and challenged with ovalbumin developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. Fisetin dose-dependently inhibited ovalbumin-induced increases in total cell count, eosinophil count, and IL-4, IL-5 and IL-13 levels recovered in bronchoalveolar lavage fluid. It attenuated ovalbumin-induced lung tissue eosinophilia and airway mucus production, mRNA expression of adhesion molecules, chitinase, IL-17, IL-33, Muc5ac and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. Fisetin blocked NF-κB subunit p65 nuclear translocation and DNA-binding activity in the nuclear extracts from lung tissues of ovalbumin-challenged mice. In normal human bronchial epithelial cells, fisetin repressed TNF-α-induced NF-κB-dependent reporter gene expression. Our findings implicate a potential therapeutic value of fisetin in the treatment of asthma through negative regulation of NF-κB pathway.

Expression and role of acidic mammalian chitinase and eotaxin-3 in chronic rhinosinusitis with nasal polyps.

OBJECTIVE: Acidic mammalian chitinase (AMCase) has been found to play an important role in allergy and asthma. Chronic rhinosinusitis with nasal polyps (CRSwNPs) is chronic inflammation with eosinophilic infiltration, which is similar to asthmatic inflammation. The role of AMCase in CRSwNPs has been less studied. Eotaxin-3 is a potent eosinophil attractant and can induce eosinophil recruitment and activation in the airways of asthmatics. The expression and role of eotaxin-3 messenger ribonucleic acid (mRNA) and its relationship to AMCase in CRSwNPs have not been studied previously. SUBJECTS AND METHODS: Twenty-three subjects with nasal polyps and nine subjects with nasal septum deviation were included in the study. The polyps and inferior turbinate mucosa were obtained as the tissue samples. Real-time reverse transcriptase-polymerase chain reaction was used to analyze and compare the expression levels of AMCase and eotaxin-3 in nasal polyps and inferior turbinate tissues (ITTs). RESULTS: AMCase and eotaxin-3 were detected in all nasal polyps and ITTs. The expression ratio of AMCase mRNA was 123.90 ± 30.60 in nasal polyps and 2.38 ± 0.41 in ITTs. The expression ratio of eotaxin-3 mRNA was 43.58 ± 15.15 and 0.84 ± 0.30, respectively. The expression of AMCase and eotaxin-3 mRNA was significantly higher in nasal polyps than in ITTs (p < .01). CONCLUSIONS: Our results reveal that AMCase and eotaxin-3 may be important mediators in the pathogenesis of nasal polyps. The increased AMCase and eotaxin-3 might lead to nasal polyp formation and growth. Future studies are needed to determine the potential of AMCase and eotaxin-3 as therapeutic targets in CRSwNPs.

Different structure and mRNA expression of Entamoeba invadens chitinases in the encystation and excystation.

Entamoeba histolytica forms chitin-walled cysts during encystation process, where formation of the cyst wall needs not only chitin synthase but also chitinase. During excystation, quadruplet amoebae emerge from the chitin-walled cysts by dissolving the wall, so that chitinase may be necessary for excystation process as well. There is, however, no report on chitinase expression during excystation. In this study, we used Entamoeba invadens, a reptilian amoeba, as a model for encystation and excystation of E. histolytica, and studied chitinase mRNA expression in those processes. Although expression of three E. invadens chitinases designated EiChit1, EiChit2, and EiChit3 during encystation has been reported, we identified another enzyme named as EiChit4 in the E. invadens genome database. Therefore, we investigated the primary structure and mRNA expression of these four chitinases of Ei in the excystation as well as the encystation by real-time reverse transcription polymerase chain reaction (RT-PCR). Like EiChit1, EiChit4 had an 8 × Cys chitin-binding domain (CBD) and a hydrophilic spacer between the CBD and catalytic domain, and was also closer to EiChit1 than EiChit2 and EiChit3 in the phylogenetic tree. During encystation, the expression of all four chitinases increased in the early phase; the increase in EiChit1 and EiChit4 was much higher than in EiChit2 and EiChit3. Then, the expression of all four chitinases sharply decreased in the later phase. In cysts, EiChit1 was most abundantly expressed and EiChit4 was at a lower level, while the expressions of EiChit2 and EiChit3 were virtually absent. Following the induction of excystation, mRNA levels of EiChit1 and EiChit4 in cysts 5 h after induction were significantly lower than those in cysts before induction, while those of EiChit2 and EiChit3 were remarkably higher than before induction. The mRNAs of only EiChit2 and EiChit3 remarkably increased when the excystation was induced in the presence of cytochalasin D. These data demonstrate different structures and expressions of four chitinases in the differentiation of E. invadens.

Up-regulation of the novel proinflammatory adipokines lipocalin-2, chitinase-3 like-1 and osteopontin as well as angiogenic-related factors in visceral adipose tissue of patients with colon cancer.

BACKGROUND: Obesity is widely recognised as an important risk factor for colorectal cancer (CC). AIM: The study aimed to evaluate the effect of CC on circulating concentrations and gene expression levels of inflammatory and angiogenesis-related factors in human visceral adipose tissue (VAT). METHODS: VAT biopsies were obtained from 18 healthy individuals and 11 patients with CC. Real-time polymerase chain reactions were performed to quantify gene expression levels and zymographic analyses were used to determine the activity of matrix metalloproteinases (MMPs). RESULTS: Patients with CC exhibited increased mRNA expression levels of lipocalin-2 (P=.014), osteopontin (P=.027), tumor necrosis factor-α (TNF-α) (P=.016) and chitinase-3 like-1 (P=.006) compared to control subjects in VAT. Gene expression levels of hypoxia-inducible factor-1 α, vascular endothelial growth factor and MMP-2 were significantly higher (P<.05) in VAT of patients with CC. The expression of insulin-like growth factor I, insulin growth factor binding protein 3 and MMP-9 followed the same trend, although no significant differences were reached. The enzymatic activity of MMP-9 was increased (P<.001) in patients with CC. Furthermore, individuals with CC showed increased (P<.05) circulating concentrations of the inflammatory markers interleukin-6, tumour necrosis factor α and hepatocyte growth factor, whereas levels of the anti-inflammatory adipokine adiponectin were decreased (P<.01). CONCLUSION: These findings represent the first observation that mRNA levels of the novel inflammatory factors lipocalin-2, chitinase-3 like-1 and osteopontin are increased in human VAT of subjects with CC. This observation together with the up-regulation of angiogenic factors suggests that adipokines secreted by VAT may be involved in the development of colon cancer.

The chitinolytic activity of Listeria monocytogenes EGD is regulated by carbohydrates but also by the virulence regulator PrfA.

Chitin, an insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), is one of the most abundant carbohydrate polymers in marine and terrestrial environments. Chitin hydrolysis by Listeria monocytogenes depends on two chitinase-encoding genes, chiA and chiB, and the aim of this study was to investigate their regulation. Chitin induces the expression of both chitinases in late exponential growth phase, and chiA but not chiB is furthermore induced by the monomer GlcNAc. Furthermore, their expression is subjected to catabolite control. Chitinases expressed by bacterial pathogens have proven to be important not only for nutrient acquisition and environmental survival but also for infecting animals and humans. Interestingly, the central L. monocytogenes virulence gene regulator, PrfA, is required for the chitinolytic phenotype, as chitinase activity was significantly reduced in prfA mutant cells compared to its level in wild-type cells. In agreement with this, Northern blot analysis showed that the amounts of chiA and chiB transcripts upon induction by chitin were significantly lower in the prfA mutant than in the wild type. The chitinolytic activity and chiA and chiB expression were reduced in the absence of the sigB gene, indicating that σ(B) is also important for the production of chitinases. The chiA, chiB, and chiA chiB mutants were not impaired for in vitro adhesion and invasion in epithelial cell lines, but the chiA chiB double mutant showed less survival ability in a chitin-enriched medium. The regulation of chitinolytic activity in L. monocytogenes is complex, and taken together, the results indicate that the biological role of this activity may not be limited to the external environment.

Chitinase production by Streptomyces sp. ANU 6277

Chitinase production by a terrestrial Streptomyces sp. ANU 6277 was studied under sub-merged fermentation. Chitinase production started after 24 h of incubation and reached maximum levels after 60 h of cultivation. A high level of chitinase activity was observed in the culture medium with pH 6 at 35ºC. Culture medium amended with 1 percent chitin was found to be suitable for maximum production of chitinase. An optimum concentration of colloidal chitin for chitinase production was determined. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that starch and yeast extract served as good carbon and nitrogen sources to enhance chitinase yield.Chitinase was purified from crude enzyme extract by single step gel filtration by Sephadex G-100. Purified chitinase of the strain exhibited a distinct protein band near 45 kDa by means of SDS-PAGE.