A Bioresponsive Genetically Encoded Antimicrobial Crystal for the Oral Treatment of Helicobacter Pylori Infection.

Helicobacter pylori (H. pylori) causes infection in the stomach and is a major factor for gastric carcinogenesis. The application of antimicrobial peptides (AMPs) as an alternative treatment to traditional antibiotics is limited by their facile degradation in the stomach, their poor penetration of the gastric mucosa, and the cost of peptide production. Here, the design and characterization of a genetically encoded H. pylori-responsive microbicidal protein crystal Cry3Aa-MIIA-AMP-P17 is described. This designed crystal exhibits preferential binding to H. pylori, and when activated, promotes the targeted release of the AMP at the H. pylori infection site. Significantly, when the activated Cry3Aa-MIIA-AMP-P17 crystals are orally delivered to infected mice, the Cry3Aa crystal framework protects its cargo AMP against degradation, resulting in enhanced in vivo efficacy against H. pylori infection. Notably, in contrast to antibiotics, treatment with the activated crystals results in minimal perturbation of the mouse gut microbiota. These results demonstrate that engineered Cry3Aa crystals can serve as an effective platform for the oral delivery of therapeutic peptides to treat gastrointestinal diseases.

Structural characterization and in vitro hepatoprotective activity of a novel antioxidant polysaccharide from fruiting bodies of the mushroom Pleurotus ferulae.

In the present study, three novel antioxidant polysaccharides (G-1, AG-1, and AG-2) were isolated and purified from Pleurotus ferulae using mouse erythrocyte hemolysis inhibitory activity as an indicator. These components showed antioxidant activity at the chemical and cellular levels. Given that G-1 displayed superior performance in protecting the human hepatocyte L02 cells against oxidative damage caused by H2O2 compared to AG-1 and AG-2 and had a higher yield and purification rate, the detailed structure of G-1 was further characterized. G-1 mainly contains six kinds of linkage type units as A: →4,6)-α-d-Glcp-(1→, B: →3)-ß-d-Glcp-(1→, C: →2,6)-ß-d-Glcp-(1→, d: ß-d-Manp(1→, E: →6)-ß-d-Galp-(1→, F: →4)-ß-d-Glcp-(1→. Finally, the potential in vitro hepatoprotective mechanism of G-1 was discussed and elucidated. Results suggested that G-1 can protect L02 cells from H2O2-induced damage by reducing the leakage of AST and ALT from the cytoplasm, enhancing the activities of SOD and CAT, and suppressing lipid peroxidation and production of LDH. G-1 could further reduce the production of ROS, stabilize mitochondrial membrane potential and maintain cell morphology. Hence, G-1 could be a valuable functional food with antioxidant and hepatoprotective activities.

New fungal protein from Pleurotus ferulae lanzi induces AMPK-mediated autophagy and G1-phase cell cycle arrest in A549 lung cancer cells.

A new protein, designated PFAP, with activity against non-small cell lung cancer (NSCLC), was isolated from Pleurotus ferulae lanzi, a medicinal and edible mushroom. The purification method involved hydrophobic interaction chromatography on a HiTrap Octyl FF column and gel filtration on a Superdex 75 column. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band with a molecular weight of 14.68 kDa. Following de novo sequencing and liquid chromatography-tandem mass spectrometry, PFAP was identified as a protein consisting of 135 amino acid residues, with a theoretical molecular weight of 14.81 kDa. Tandem mass tag (TMT)™-based quantitative proteomic analysis and western blotting revealed that AMP-activated protein kinase (AMPK) was significantly upregulated in NSCLC A549 cells, following PFAP treatment. The downstream regulatory factor mammalian target of rapamycin (mTOR) was suppressed, resulting in the activation of autophagy and upregulated expressions of P62, LC3 II/I, and other related proteins. PFAP blocked NSCLC A549 cells in the G1 phase of the cell cycle via upregulating P53 and P21, while subsequently downregulating the expression of cyclin-dependent kinases. PFAP suppresses tumour growth via the same mechanism in a xenograft mouse model in vivo. These results demonstrate that PFAP is a multifunctional protein with anti-NSCLC properties.

A novel mushroom (Auricularia polytricha) glycoprotein protects against lead-induced hepatoxicity, promotes lead adsorption, inhibits organ accumulation of lead, upregulates detoxifying proteins, and enhances immunoregulation in rats.

Introduction: Lead is a ubiquitous environmental and industrial pollutant. Its nonbiodegradable toxicity induces a plethora of human diseases. A novel bioactive glycoprotein containing 1.15% carbohydrate, with the ability of adsorbing lead and effecting detoxification, has been purified from Auricularia polytricha and designated as APL. Besides, its mechanisms related to regulation of hepatic metabolic derangements at the proteome level were analyzed in this study. Methods: Chromatographic techniques were utilized to purify APL in the current study. For investigating the protective effects of APL, Sprague-Dawley rats were given daily intraperitoneal injections of lead acetate for establishment of an animal model, and different dosages of APL were gastrically irrigated for study of protection from lead detoxification. Liver samples were prepared for proteomic analyses to explore the detoxification mechanisms. Results and discussion: The detoxifying glycoprotein APL displayed unique molecular properties with molecular weight of 252-kDa, was isolated from fruiting bodies of the edible fungus A. polytricha. The serum concentrations of lead and the liver function biomarkers aspartate and alanine aminotransferases were significantly (p<0.05) improved after APL treatment, as well as following treatment with the positive control EDTA (300 mg/kg body weight). Likewise, results on lead residue showed that the clearance ratios of the liver and kidneys were respectively 44.5% and 18.1% at the dosage of APL 160 mg/kg, which was even better than the corresponding data for EDTA. Proteomics disclosed that 351 proteins were differentially expressed following lead exposure and the expression levels of 41 proteins enriched in pathways mainly involved in cell detoxification and immune regulation were normalized after treatment with APL-H. The results signify that APL ameliorates lead-induced hepatic injury by positive regulation of immune processing, and suggest that APL can be applied as a therapeutic intervention of lead poisoning in clinical practice. This report represents the first demonstration of the protective action of a novel mushroom protein on lead-elicited hepatic toxicity.

Fungal antitumor protein D1 is internalized via endocytosis and inhibits non-small cell lung cancer proliferation through MAPK signaling pathway.

Lung cancer has the highest mortality among cancer-related deaths worldwide. Among lung cancers, non-small cell lung cancer (NSCLC) is the most common histological type. In the previous research, we isolated a protein (D1) from Boletus bicolor that inhibits the proliferation of NSCLC cell lines. In this study, we elucidated the internalization mechanism and antitumor mechanism of protein D1 in A549 cells. Protein D1 has a strong inhibitory effect on A549 cells. It binds to secretory carrier membrane protein 3 on the A549 cell membrane and enters A549 cells by clathrin-mediated endocytosis. In vitro, protein D1 activates mitogen-activated protein kinase (MAPK) signaling pathway. JNK and p38MAPK are the biological targets for protein D1. In vivo, protein D1 inhibits the tumor growth of NSCLC xenografts by inducing apoptosis and inhibiting cell proliferation. Protein D1 alters the expression of genes related to apoptosis, cell cycle, and MAPK signaling pathway in tumor cells.

Aberrant Transferrin and Ferritin Upregulation Elicits Iron Accumulation and Oxidative Inflammaging Causing Ferroptosis and Undermines Estradiol Biosynthesis in Aging Rat Ovaries by Upregulating NF-Κb-Activated Inducible Nitric Oxide Synthase: First Demonstration of an Intricate Mechanism.

We report herein a novel mechanism, unraveled by proteomics and validated by in vitro and in vivo studies, of the aberrant aging-associated upregulation of ovarian transferrin and ferritin in rat ovaries. The ovarian mass and serum estradiol titer plummeted while the ovarian labile ferrous iron and total iron levels escalated with age in rats. Oxidative stress markers, such as nitrite/nitrate, 3-nitrotyrosine, and 4-hydroxy-2-nonenal, accumulated in the aging ovaries due to an aberrant upregulation of the ovarian transferrin, ferritin light/heavy chains, and iron regulatory protein 2(IRP2)-mediated transferrin receptor 1 (TfR1). Ferritin inhibited estradiol biosynthesis in ovarian granulosa cells in vitro via the upregulation of a nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and p65/p50-induced oxidative and inflammatory factor inducible nitric oxide synthase (iNOS). An in vivo study demonstrated how the age-associated activation of NF-κB induced the upregulation of iNOS and the tumor necrosis factor α (TNFα). The downregulation of the keap1-mediated nuclear factor erythroid 2-related factor 2 (Nrf2), that induced a decrease in glutathione peroxidase 4 (GPX4), was observed. The aberrant transferrin and ferritin upregulation triggered an iron accumulation via the upregulation of an IRP2-induced TfR1. This culminates in NF-κB-iNOS-mediated ovarian oxi-inflamm-aging and serum estradiol decrement in naturally aging rats. The iron accumulation and the effect on ferroptosis-related proteins including the GPX4, TfR1, Nrf2, Keap1, and ferritin heavy chain, as in testicular ferroptosis, indicated the triggering of ferroptosis. In young rats, an intraovarian injection of an adenovirus, which expressed iron regulatory proteins, upregulated the ovarian NF-κB/iNOS and downregulated the GPX4. These novel findings have contributed to a prompt translational research on the ovarian aging-associated iron metabolism and aging-associated ovarian diseases.

An anti-inflammatory and anti-fibrotic proprietary Chinese medicine nasal spray designated as Allergic Rhinitis Nose Drops (ARND) with potential to prevent SARS-CoV-2 coronavirus infection by targeting RBD (Delta)- angiotensin converting enzyme 2 (ACE2) binding.

BACKGROUND: Since the outbreak of COVID-19 has resulted in over 313,000,000 confirmed cases of infection and over 5,500,000 deaths, substantial research work has been conducted to discover agents/ vaccines against COVID-19. Undesired adverse effects were observed in clinical practice and common vaccines do not protect the nasal tissue. An increasing volume of direct evidence based on clinical studies of traditional Chinese medicines (TCM) in the treatment of COVID-19 has been reported. However, the safe anti-inflammatory and anti-fibrotic proprietary Chinese medicines nasal spray, designated as Allergic Rhinitis Nose Drops (ARND), and its potential of re-purposing for suppressing viral infection via SARS-CoV-2 RBD (Delta)- angiotensin converting enzyme 2 (ACE2) binding have not been elucidated. PURPOSE: To characterize ARND as a potential SARS-CoV-2 entry inhibitor for its possible preventive application in anti-virus hygienic agent. METHODS: Network pharmacology analysis of ARND was adopted to asacertain gene targets which were commonly affected by COVID-19. The inhibitory effect of ARND on viral infection was determined by an in vitro pseudovirus assay. Furthermore, ARND was confirmed to have a strong binding affinity with ACE2 and SARS-CoV-2 spike-RBD (Delta) by ELISA. Finally, inflammatory and fibrotic cell models were used in conjunction in this study. RESULTS: The results suggested ARND not only inhibited pseudovirus infection and undermined the binding affinity between ACE2 and the Spike protein (Delta), but also attenuated the inflammatory response upon infection and may lead to a better prognosis with a lower risk of pulmonary fibrosis. The data in this study also provide a basis for further development of ARND as an antiviral hygienic product and further investigations on ARND in the live virus, in vivo and COVID-19 patients. ARND holds promise for use in the current COVID-19 outbreak as well as in future pandemics. CONCLUSION: ARND could be considered as a safe anti-SARS-CoV-2 agent with potential to prevent SARS-CoV-2 coronavirus infection.

An Update of Lectins from Marine Organisms: Characterization, Extraction Methodology, and Potential Biofunctional Applications.

Lectins are a unique group of nonimmune carbohydrate-binding proteins or glycoproteins that exhibit specific and reversible carbohydrate-binding activity in a non-catalytic manner. Lectins have diverse sources and are classified according to their origins, such as plant lectins, animal lectins, and fish lectins. Marine organisms including fish, crustaceans, and mollusks produce a myriad of lectins, including rhamnose binding lectins (RBL), fucose-binding lectins (FTL), mannose-binding lectin, galectins, galactose binding lectins, and C-type lectins. The widely used method of extracting lectins from marine samples is a simple two-step process employing a polar salt solution and purification by column chromatography. Lectins exert several immunomodulatory functions, including pathogen recognition, inflammatory reactions, participating in various hemocyte functions (e.g., agglutination), phagocytic reactions, among others. Lectins can also control cell proliferation, protein folding, RNA splicing, and trafficking of molecules. Due to their reported biological and pharmaceutical activities, lectins have attracted the attention of scientists and industries (i.e., food, biomedical, and pharmaceutical industries). Therefore, this review aims to update current information on lectins from marine organisms, their characterization, extraction, and biofunctionalities.

Considerations regarding affinity determinants for aflatoxin B1 in binding cavity of fungal laccase based on in silico mutational and in vitro verification studies.

Laccase, a multicopper oxidase, is well known for its industrial potentials to remove environmental pollutants due to its low substrate specificity to oxidize phenols and thus catalytic versatility. Many efforts focused on the metabolic mechanism, yet to decipher the structural determinants responsible for the differentiation between substrates. Aflatoxin B1 (AFB1), a new substrate for laccase, is a mycotoxin with a formidable environmental threat to public health and food safety. In the present study, we combined biochemical, in silico mutational and molecular-docking data to gain an insight to the function of key residues in the active cavity close to the T1 copper site in a characterized recombinant laccase from Cerrena unicolor (rCuL). Kinetic data for computer-assisted virtual mutants established the binding affinity of hydrogen bonds and residues (Asn336, Asp207, Val391, and Thr165) in rCuL to AFB1. The augmented binding affinity to AFB1 may be related to the conformational rearrangements of the laccase and its ability to hydrogen-bond with the substrate. Furthermore, the optimal pH and temperature for rCuL and variants mediated AFB1 degradation may depend on their pH stability and thermostability. Our findings reinforce the importance of the structure-function relationship of fungal laccases in degrading AFB1, providing mechanistic guidance for future biocatalyst and bioengineering applications.

Lycium barbarum polysaccharide attenuates emotional injury of offspring elicited by prenatal chronic stress in rats via regulation of gut microbiota.

Stress during pregnancy is not only detrimental to a woman's own physical and mental health, but can also cause changes in the intrauterine environment and even have an impact on later growth and development, this study was designed to understand the changes of gut microbiota in the maternal and offspring caused by prenatal chronic stress, and to explore the regulatory effect of LBP on gut microbiota, and then to improve the emotional damage caused by prenatal chronic stress in the offspring. A rat model of prenatal chronic stress was made and used LBP to intervene by gavage. Fresh feces of offspring were collected, the concentration of microbial metabolites were tested by ELISA. Illumina MiSeqPE300 sequencing technology was used to determine the sequence of 16S rRNA V3-V4 of microorganisms. On the PND 42, the emotional function of offspring were tested by open-field test (OFT), sucrose preference test (SPT) and tail of suspend test (TST). Results indicated that stress factors increased the plasma corticosterone level of rats during pregnancy and they appeared depressive behaviors. The body weight of offspring during prenatal chronic stress was lower than the control group, and the plasma corticosterone level was increased. Prenatal chronic stress had a significant impact on emotional performance of the offspring on OFT, SPT and TST. Alpha diversity of gut microbiota and microbiota composition in offspring of prenatal chronic stress was attenuated and some relationships existed between these parameters. LBP treatment reduced offspring's plasma corticosterone level and improved their body weight, changed the emotional function, increased the diversity of gut microbiota. Collectively, these findings disclose that prenatal chronic stress not only causes emotional injury on the offspring, but also changes the gut microbiota of the mother and offspring; LBP may regulate the intestinal flora of the mother, then reducing the influence of stress factors on the emotional injury of offspring.

Interrelationship among paraptosis, apoptosis and autophagy in lung cancer A549 cells induced by BEAP, an antitumor protein isolated from the edible porcini mushroom Boletus edulis.

In today's world, cancer is still the leading cause of human death. Among them, the incidence and mortality of lung cancer remain high, and have become the focus of research in the world. BEAP, a protein with anti-lung cancer activity, was isolated and purified from the edible mushroom Boletus edulis. Previous studies have shown that BEAP can inhibit the proliferation of non-small cell lung cancer A549 cells by inducing apoptosis and cell cycle arrest in vitro and in vivo. However, there are many ways in which antitumor proteins from edible and medicinal mushroom play their roles. It is worth exploring whether there are other antitumor mechanisms of BEAP, which can provide reference value for the development of new drugs targeting non-small cell lung cancer and the repurposing of existing drugs.

Purified antioxidant from the medicinal mushroom Phellinus pini protects rat H9c2 cell against H2 O2 -induced oxidative stress.

In this study, through the combination of AB-8 macroporous resin, Sephadex LH-20 column chromatography and semi-preparative HPLC, an antioxidant component was purified from the crude extract of Phellinus pini, thereby evaluating the cardioprotective effect of the fraction. As a result, total phenolic content of the 60% ethanol elution was increased by 4.8-fold after one run treatment on Sephadex LH-20 chromatography with gradient elution. After semi-preparative HPLC separation, the first peak (PP-S4-1) showed that inhibition ratio of erythrocyte hemolysis was 91.9%, and inhibition ratio of lipid peroxidation was also increased by 87.6%, at 50 µg/ml (p < .01). Based on the results of ESI-MS, 1 HNMR, 13 CNMR, and RP-HPLC compared to many published results, PP-S4-1was identified as catechin (MW 290.015, C15 H14 O6 ). The results showed that PP-S4-1 pretreatment made cell viability increased, and the generation of reactive oxygen species (ROS) inhibited. Meanwhile, PP-S4-1 remarkably decreased the fluorescence intensity of Ca2+ , and increased mitochondrial membrane potential (MMP; ΔΨm). In addition, PP-S4-1 could significantly inhibit the decrease of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity as well as the increase of MDA content in H9c2 cells induced by H2 O2 . Moreover, pretreatment with PP-S4-1 significantly improved the morphological changes and prevented H2 O2 -induced DNA damage. Therefore, this study clarifies the ability of PP-S4-1 to treat H9c2 cell oxidative stress damage induced by H2 O2 through its antioxidant effect. PRACTICAL APPLICATIONS: This research is not only helpful to elaborate the cardioprotective effect of Phellinus pini but also can contribute to the development of health foods or drug supplements for heart disease in the future. This is the first report dealing with phenolic component and cardioprotective activity of a medicinal mushroom P. pini belonging to the genus Phellinus.

Coating shiitake mushrooms (Lentinus edodes) with a polysaccharide from Oudemansiella radicata improves product quality and flavor during postharvest storage.

In this work, we investigated whether coating fresh shiitake mushrooms with a polysaccharide isolated from Oudemansiella radicata (ORWP) would impact key quality characteristics after 18 d of storage at 4 °C. We found that ORWP-coated mushrooms had significant improvements in many qualities during storage, including reduced weight loss, improved firmness, reduced browning, decreased malondialdehyde content, and an improved physical microstructure. Further, ORWP-coated mushrooms had higher contents of nutritional and cell wall compounds compared to control samples. ORWP-coated mushrooms had reduced activities of the following enzymes: protease, polyphenol oxidase, peroxidase, phenylalanine ammonia lyase, cellulase, and chitinase, relative to control samples. However, mushrooms coated with ORWP had higher concentrations of superoxide dismutase and catalase, as well as higher contents of certain key monosodium glutamate-resembling amino acids, umami 5'-nucleotides and 1-octen-3-ol. These findings suggest that ORWP coatings have potential value as a method to improve the postharvest quality of shiitake mushrooms.

Autophagy signals orchestrate chemoresistance of gynecological cancers.

Gynecological cancers are characterized by a high mortality rate when chemoresistance develops. Autophagy collaborates with apoptosis and participates in homeostasis of chemoresistance. Recent findings supported that crosstalk of necrotic, apoptotic and autophagic factors, and chemotherapy-driven hypoxia, oxidative stress and ER stress play critical roles in chemoresistance in gynecological cancers. Meanwhile, current studies have shown that autophagy could be regulated by and cooperate with metabolic regulator, survival factors, stemness factors and specific post-translation modification in chemoresistant tumor cells. Meanwhile, non-coding RNA and autophagy crosstalk also contribute to the chemoresistance. Until now, analysis of individual autophagy factors towards the clinical significance and chemoresistance in gynecological cancer is still lacking. We suggest comprehensive integrated analysis of cellular homeostasis and tumor microenvironment to clarify the role of autophagy and the associated factors in cancer progression and chemoresistance. Panel screening of pan-autophagic factors will pioneer the development of risk models for predicting efficacy of chemotherapy and guidelines for systematic treatment and precision medicine.

Study on the Biocontrol Potential of Antifungal Peptides Produced by Bacillus velezensis against Fusarium solani That Infects the Passion Fruit Passiflora edulis.

A bacterium identified as Bacillus velezensis with a growth inhibitory effect against Fusarium solani, a pathogen that caused basal stem rot in the passion fruit Passiflora edulis, was isolated in this study. From the fermentation broth of B. velezensis, a type of antifungal peptide (named BVAP) with a molecular weight of ca. 1.5 kDa was purified and found to be fengycin. BVAP suppressed mycelial growth in F. solani with an IC50 of 5.58 µg/mL, which was superior to those of the chemical fungicides thiram (41.24 µg/mL) and hymexazol (343.31 µg/mL). The antifungal activity remained stable after exposure to 50-100 °C or following incubation with solutions at pH 1-3. Further research revealed that BVAP increased the permeability of the F. solani mycelial membrane, brought about swelling at the tips of hyphae, and elicited abnormal accumulation of nucleic acids and chitin at the sites of swelling. These findings indicate that BVAP possessed a remarkable biocontrol potential toward F. solani.

The mechanism of enriched environment repairing the learning and memory impairment in offspring of prenatal stress by regulating the expression of activity-regulated cytoskeletal-associated and insulin-like growth factor-2 in hippocampus.

BACKGROUND: Prenatal stress can cause neurobiological and behavioral defects in offspring; environmental factors play a crucial role in regulating the development of brain and behavioral; this study was designed to test and verify whether an enriched environment can repair learning and memory impairment in offspring rats induced by prenatal stress and to explore its mechanism involving the expression of insulin-like growth factor-2 (IGF-2) and activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus of the offspring. METHODS: Rats were selected to establish a chronic unpredictable mild stress (CUMS) model during pregnancy. Offspring were weaned on 21st day and housed under either standard or an enriched environment. The learning and memory ability were tested using Morris water maze and Y-maze. The expression of IGF-2 and Arc mRNA and protein were respectively measured by using RT-PCR and Western blotting. RESULTS: There was an elevation in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy. Maternal stress's offspring exposed to an enriched environment could decrease their plasma corticosterone level and improve their weight. The offspring of maternal stress during pregnancy exhibited abnormalities in Morris water maze and Y-maze, which were improved in an enriched environment. The expression of IGF-2, Arc mRNA, and protein in offspring of maternal stress during pregnancy was boosted and some relationships existed between these parameters after being exposed enriched environment. CONCLUSIONS: The learning and memory impairment in offspring of prenatal stress can be rectified by the enriched environment, the mechanism of which is related to the decreasing plasma corticosterone and increasing hippocampal IGF-2 and Arc of offspring rats following maternal chronic stress during pregnancy.

Purification and characterization of a novel protein with activity against non-small-cell lung cancer in vitro and in vivo from the edible mushroom Boletus edulis.

A new anti-tumor protein (designated as Boletus edulis or in short BEAP) was isolated from dried fruit bodies of the edible bolete mushroom Boletus edulis. The purification protocol employed comprised fast ion exchange chromatography on a Hitrap Q column and ion exchange chromatography on a DEAE-52 cellulose column. Superdex G75 gel filtration and SDS-PAGE analysis revealed that BEAP was a protein with a molecular weight of 16.7 KD. The protein exhibited potent anti-cancer activity on A549 cells both in vitro and in vivo. With the use of AO/EB staining, annexin V-FITC/PI, and Western blotting, it was demonstrated in vitro that the cytotoxicity of BEAP was mediated by induction of apoptosis and arrest of A549 cells in the G1 phase of the cell cycle. BEAP significantly suppressed the growth of A549 solid tumors in vivo. These results prove that BEAP is a new multifunctional protein with anti-tumor and anti-metastasis capabilities.

A thermophilic chitinase 1602 from the marine bacterium Microbulbifer sp. BN3 and its high-level expression in Pichia pastoris.

Chitinases play an important role in many industrial processes, including the preparation of oligosaccharides with potential applications. In the present study, a 1,713 bp gene of Chi1602, derived from a marine bacterium Microbulbifer sp. BN3, encoding a GH18 family chitinase, was expressed at high levels in Pichia pastoris. Distinct from most of the marine chitinases, the recombinant chitinase 1602 exhibited maximal activity at 60 °C and over a broad pH range between 5.0 and 9.0, and was stable at 50 °C and over the pH range 4.0-9.0. The hydrolytic products derived from colloidal chitins comprised mainly (GlcNAc)2 and GlcNAc, indicating that rChi1602 is a GH18 processive chitinase in conformity with its hypothetical structure. However, rChi1602 showed traces of ß-N-acetylglucosaminidase activity on substrates such as powder chitin, chitosan, and ethylene glycol chitin. The thermophilic rChi1602, which manifests adaptation to a wide pH range and can be expressed at a high level in P. pastoris, is advantageous for applications in industrial processes.

Therapeutic potential of a novel prodrug of green tea extract in induction of apoptosis via ERK/JNK and Akt signaling pathway in human endometrial cancer.

BACKGROUND: Previous studies have shown a major green tea polyphenol (-)-epigallocatechin-3-gallate ((-)-EGCG) as a powerful anti-cancer agent. However, its poor bioavailability and requirement of a high dosage to manifest activity have restricted its clinical application. Recently, our team synthesized a peracetate-protected derivative of EGCG, which can act as a prodrug of (-)-EGCG (ProEGCG) with enhanced stability and improved bioavailability in vitro and in vivo. Herein, we tested the therapeutic efficacy of this novel ProEGCG, in comparison to EGCG, toward human endometrial cancer (EC). METHODS: In this study, the effects of ProEGCG and EGCG treatments on cell growth, cell survival and modulation of intracellular signaling pathways in RL95-2 and AN3 CA EC cells were compared. The antiproliferative effect was evaluated by cell viability assay. Apoptosis was measured by annexin/propidium iodide staining. Expression of mitogen-activated protein kinases, markers of proliferation and apoptosis were measured by immunoblot analysis. In addition, the effects of ProEGCG and EGCG on tumor growth, vessel formation and gene expression profiles on xenograft models of the EC cells were investigated. RESULTS: We found that treatment with ProEGCG, but not EGCG, inhibited, in a time- and dose-dependent manner, the proliferation and increased apoptosis of EC cells. Treatment with low-dose ProEGCG significantly enhanced phosphorylation of JNK and p38 MAPK and inhibited phosphorylation of Akt and ERK which are critical mediators of apoptosis. ProEGCG, but not EGCG, elicited a significant decrease in the growth of the EC xenografts, promoted apoptotic activity of tumour cells in the EC xenografts, and decreased microvessel formation, by differentially suppressing anti-apoptotic molecules, NOD1 and NAIP. Notably, no obvious adverse effects were detected. CONCLUSIONS: Taken together, ProEGCG at a low dose exhibited anticancer activity in EC cells through its anti-proliferative, pro-apoptotic and anti-tumor actions on endometrial cancer in vitro and in vivo. In contrast, a low dose of EGCG did not bring about similar effects. Importantly, our data demonstrated the efficacy and safety of ProEGCG which manifests the potential of a novel anticancer agent for the management of endometrial cancer.

A novel antitumor protein from the mushroom Pholiota nameko induces apoptosis of human breast adenocarcinoma MCF-7 cells in vivo and modulates cytokine secretion in mice bearing MCF-7 xenografts.

Breast cancer is the most common malignant tumor in women and one of the three most common cancers worldwide. It is a life-threatening disease among women and the leading cause of death among women. New drugs or new drug translations and laboratory clinical studies are ongoing. A new antitumor protein (PNAP) purified from edible fungus Pholiota nameko has potential for treating breast cancer. We have previously found that PNAP exhibits anti-proliferative and apoptosis-inducing activities in a human breast cancer cell line (MCF-7). In this study, we constructed a BALB/c mouse model of MCF-7 tumor xenografts. In vivo experiments show that PNAP can effectively inhibit the malignant proliferation of MCF-7 solid tumors. This is because PNAP can successfully activate the death receptor pathway and mitochondrial apoptosis pathway of MCF-7 tumor cells in vivo, and induce tumor cells to wither. It is estimated that PNAP may also have an immunoregulatory ability to indirectly inhibit malignant proliferation of tumors. We also found that PNAP may also have the immunomodulatory ability to indirectly inhibit the malignant proliferation of tumors, which can shift the balance of Th1/Th2 to Th1 and eventually inhibit the growth of tumors. The study reveals a new therapeutic approach for breast cancer patient.