Heterologous expression of pediocin PA-1 in Pichia pastoris: cloning, expression, characterization, and application in pork bologna preservation.

OBJECTIVE: Pediocin PA-1, an antimicrobial peptide derived from Pediococcus acidilactici PAC1.0, has a potential application as a food preservative thanks to its strong inhibitory activity against the foodborne pathogen L. monocytogenes. This study aimed to produce Pediocin PA-1 from the yeast P. pastoris and evaluate its characteristics. METHODS: Gene encoding Pediocin PA-1 was integrated into P. pastoris X33 genome to establish the strain X33::ped, which could produce and secrete this peptide into culture medium. The antimicrobial activity of Pediocin PA-1 was examined using agar diffusion assay. The stability of pediocin PA-1 was determined based on its remaining antibacterial activity after exposure to proteases and extreme pH and temperatures. The potential use of this bacteriocin in food preservation was demonstrated using the L. monocytogenes infected pork bologna. The anticancer activity of Pediocin PA-1 was also investigated on some cancer cells using MTT assay. RESULTS: We established the yeast P. pastoris X33::ped capable of producing pediocin PA-1 with antimicrobial activity against L. monocytogenes and some other harmful bacteria. Pediocin PA-1 was stable at 100˚C and resistant against pH 1-12 for 1 h, but susceptible to trypsin, α-chymotrypsin, and proteinase K. This peptide could reduce the number of L. monocytogenes in pork bologna by 3.59 log CFU/g after 7 days of storage at 4˚C. Finally, Pediocin PA-1 (25 µg/ml) inhibited the proliferation of A549 and Hela cancer cells. CONCLUSION: We succeeded in producing active Pediocin PA-1 from P. pastoris and demonstrated its potential use in food preservation and pharmaceutical industry.

KTt-45, a T-type calcium channel blocker, acts as an anticancer agent by inducing apoptosis on HeLa cervical cancer cell line.

The abnormal expression in the T-type calcium channels is involved in various cancer types, thus inhibiting T-type calcium channels is one of approaches in cancer treatment. The fact that KTt-45 acted as a T-type calcium channel inhibitor as well as a pain-relief agent prompts us to address if KTt-45 plays any role against cancer cells. The results showed that KTt-45 caused cytotoxic effects towards HeLa cervical, Raji lymphoma, MCF-7 breast cancer, and A549 lung cancer cell lines with IC50 values less than 100 µM, in which highly selective toxicity was against HeLa cells (IC50 = 37.4 µM, SI > 3.2). Strikingly, the KTt-45 induced an accumulation of cytoplasmic vacuoles after 48 h treatment and mitochondrial-dependent apoptosis activation as evidenced by morphological features, chromatin condensation, nuclear fragmentation, and significant activation of caspase-9 as well as caspase-3. In conclusion, KTt-45 could inhibit cell growth and trigger mitochondrial-dependent apoptosis in HeLa cervical cancer cells. The results, taken together, strongly demonstrated that KTt-45 is a potential agent for further study on anticancer drug development which not only targets cancer cells but also helps to relieve neuropathic pain in cancer patients.

Rumdul (Sphaerocoryne affinis) Antioxidant Activity and Its Potential for Parkinson's Disease Treatment.

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by progressive deterioration of motor function and loss of dopaminergic neurons in the substantia nigra. Although PD is more common in people over 60 years old, people with young-onset PD tend to increase recently. Up to now, there is no cure for PD; therapies mainly focus on reducing symptoms and improving patient quality of life. Thus, the requirement of exploring new medications is needed. There is a strong relationship between oxidative stress and PD. Therefore, antioxidant compounds have been considered as a novel therapy for PD. In this study, we indicated a new potential candidate for PD treatment, rumdul fruit (Sphaerocoryne affinis-a member of the Annonaceae family), due to evaluating its activities on the fly model of Parkinson. Our experimental results showed that rumdul fruit water extract (RFWE) has a strong antioxidant capacity with IC50 value in DPPH assay which was 85.62 ± 1.05 µg/mL. The use of RFWE at concentrations of 3, 6, and 12 mg/mL could strongly ameliorate the locomotor disabilities and dopaminergic neuron degeneration. Although the RFWE at high concentrations like 12 mg/mL and 18 mg/mL could induce some side effects on fly development and viability, our data strongly demonstrated that RFWE effectively rescued PD phenotypes on the fly model. Although component in the plant extract, as well as the molecular mechanism helping to recover the phenotype, has not been elucidated yet, the research contributed strong scientific evidence for further research on applying rumdul as a novel natural source for PD treatment.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli.

BACKGROUND: The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimulating Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. METHODS: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the conjugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. RESULTS: PEGylated GCSF was obtained with high purity (∼97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). CONCLUSION: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

Ethyl acetate extract of Elephantopus mollis Kunth induces apoptosis in human gastric cancer cells.

BACKGROUND: Gastric cancer is one of the most leading causes of cancer death worldwide. Therefore, treatment studies have been being conducted, one of which is screening of novel agents from medicinal herbs. Elephantopus mollis Kunth (EM) belonging to Asteraceae family is a perennial herb with several therapeutic properties including anticancer activity. However, the effect of this species on gastric cancer has not been reported yet. In this study, cytotoxicity of different EM crude extracts was investigated on AGS gastric cancer cell line. Besides, the effects of extract on nuclear morphology, caspase-3 activation, and gene expression were also explored. RESULTS: The results showed that ethyl acetate extract exhibited a remarkably inhibitory ability (IC50 = 27.5 µg/ml) on the growth of AGS cells, while causing less toxicity to normal human fibroblasts. The extract also induced apoptotic deaths in AGS cells as evidenced by cell shrinkage, formation of apoptotic bodies, nuclear fragmentation, caspase-3 activation, and the upregulation of BAK and APAF-1 pro-apoptotic genes related to mitochondrial signaling pathway. Specifically, BAK and APAF-1 mRNA expression levels showed 2.57 and 2.71-fold increases respectively. CONCLUSIONS: The current study not only proved the anti-gastric cancer activity of EM ethyl acetate extract but also proposed its molecular mechanism. The extract could be a potential candidate for further investigation.

Yeast cell surface displaying VP28 antigen and its potential application for shrimp farming.

VP28 is an envelope protein of White Spot Syndrome Virus (WSSV), which has been shown in previous studies to induce a high immune response in shrimp. VP28 has been produced in some host systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris as free protein. Here we showed a new strategy of anchoring VP28 on the Saccharomyces cerevisiae yeast surface and using the yeast cell extract combined with probiotic as an oral vaccine for shrimp farming. We have successfully constructed a recombinant yeast cell capable of expressing VP28 on the cell surface. The feeding diet combined with VP28 anchored yeast cell extract provided significant assurance to Litopenaeus vannamei, challenged by WSSV, resulting in a relative percent survival (RPS) of 87.10 ± 2.15%. Interestingly, the utilization of VP28 anchored yeast cell extract could enhance the efficiency of probiotic strains like Lactobacillus and Bacillus on shrimp farming. The results in both laboratory scales and field trials using extract of VP28 displaying Saccharomyces showed a growth-promoting effect in shrimp, assessed through average shrimp weight. Taken together, our results in this study demonstrated a new successful strategy of using yeast cell surface as a tool to produce VP28-based oral vaccine for shrimp aquaculture. KEY POINTS: • A new strategy of using VP28 antigen as anchored protein on S. cerevisiae yeast cell surface (S. cerevisiae::VP28) • The utilization of VP28 antigen and yeast as S. cerevisiae::VP28 extract enhanced potential protection of Litopenaeus vannamei against White Spot Syndrome Virus (RPS 87.10%) • The use of S. cerevisiae::VP28 extract increased efficiency of probiotic on shrimp growth-promoting effect either lab-scale or field trial.

Production of recombinant human G-CSF from non-classical inclusion bodies in Escherichia coli.

Recombinant granulocyte colony-stimulating factor (G-CSF) protein produced in Escherichia coli has been widely used for the treatment of neutropenia induced by chemotherapy for decades. In E. coli cells, G-CSF is usually expressed as inactive inclusion bodies, which requires costly and inefficient denaturation and refolding steps to obtain the protein in its active form. However, following the findings of previous studies, we here successfully produced G-CSF in E. coli as non-classical inclusion bodies (ncIBs), which contained likely correctly folded protein. The ncIBs were easily dissolved in 0.2% N-lauroylsarcosine solution and then directly applied to a Ni-NTA affinity chromatography column to get G-CSF with high purity (> 90%). The obtained G-CSF was demonstrated to have a similar bioactivity with the well-known G-CSF containing product Neupogen (Amgen, Switzerland). Our finding clearly verified that the G-CSF production from ncIBs is a feasible approach to improve the yield and lower the cost of G-CSF manufacturing process.

Wound Healing Activity of Crassocephalum crepidioides (Benth.) S. Moore. Leaf Hydroethanolic Extract.

Crassocephalum crepidioides (Benth.) S. Moore. has been used to treat small wounds by minority people in Lam Dong, Vietnam. However, there has been no scientific evidences about its wound healing activity. This study is aimed at evaluating the wound healing activity of Crassocephalum crepidioides hydroethanolic extract via its antioxidant and anti-inflammation activities and healing capability on a mouse excision wound model. Crassocephalum crepidioides hydroethanolic extract (CCLE) at a dose of 50 mg/kg/day reduced the wound closure time about 3.5 days, compared to vehicle treatment. The granulation tissue on day 7 after surgery from the treated group showed a 2.8-fold decrease in the density of inflammatory cells, 1.9-fold increase in the fibroblast density, and a higher number of blood vessels. Real-time PCR analysis indicated that the mRNA expression level of NF-κB1 and TNF-α mRNA in CCLE-treated wounds decreased by 4.6 and 3.3 times, respectively, while TGF-ß1 and VEGF were found to increase by 3.3 and 2.4 times, respectively. Our experimental data provided proofs of Crassocephalum crepidioides leaf wound healing activity due to its antioxidant, anti-inflammation, fibroblast proliferation, wound contraction, and angiogenesis effects.

Targeting UCH in Drosophila melanogaster as a model for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease caused by genetic or environmental factors. Among several animal models, the Drosophila melanogaster is one of the valuable models  widely used in studying genes and proteins implicated in PD. UCH-L1 (Ubiquitin carboxyl-terminal hydrolase L1) which is involved in formation of Lewy bodies, shows loss of function mutations in PD causing degeneration of dopaminergic neurons in mice. Here, we summarize the results from studying the UCH-L1 and its knockdown in Drosophila model of PD with respect to movement, degeneration of dopamine producing neurons, dopamine deficiency and age dependent dependency of progression of the disease.  The knockdown of the UCH-L1 in Drosophila can be used in studying the epidemiology of the disease as well as in drug screening for finding therapeutic targets for PD.

Parkinson's Disease Model.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. It is known that there are many factors, either genetic or environmental factors, involved in PD, but the mechanism of PD is still not fully understood. Several animal models have been established to study the mechanisms of PD. Among these models, Drosophila melanogaster has been utilized as a valuable model to get insight into important features of PD. Drosophila melanogaster possesses a well-developed dopaminergic (DA) neuron system which is known to play an important role in PD pathogenesis. The well understanding of DA neurons from early larval through adult stage makes Drosophila as a powerful model for investigating the progressive neurodegeneration in PD. Besides, the short life cycle of Drosophila melanogaster serves an advantage in studying epidemiological features of PD. Most of PD symptoms can be mimicked in Drosophila model such as progressive impairment in locomotion, DA neuron degeneration, and some other non-motor symptoms. The Drosophila models of PD, therefore, show a great potential in application for PD genetic and drug screening.

Neuron-specific knockdown of Drosophila PDHB induces reduction of lifespan, deficient locomotive ability, abnormal morphology of motor neuron terminals and photoreceptor axon targeting.

Pyruvate dehydrogenase complex deficiency (PDCD) is a common primary cause of defects in mitochondrial function and also can lead to peripheral neuropathy. Pyruvate dehydrogenase E1 component subunit beta (PDHB) is a subunit of pyruvate dehydrogenase E1, which is a well-known component of PDC. In Drosophila melanogaster, the CG11876 (dPDHB) gene is a homolog of human PDHB. In this study, we established a Drosophila model with neuron-specific knockdown of dPDHB to investigate its role in neuropathy pathogenesis. Knockdown of dPDHB in pan-neurons induced locomotor defects in both larval and adult stages, which were consistent with abnormal morphology of the motor neuron terminals at neuromuscular junctions and mitochondrial fragmentation in brains. Moreover, neuron-specific knockdown of dPDHB also shortened the lifespan of adult flies. In addition, flies with knockdown of dPDHB manifested a rough eye phenotype and aberrant photoreceptor axon targeting. These results with the Drosophila model suggest the involvement of PDHB in peripheral neuropathy.

Ubiquitin carboxyl hydrolase L1 significance for human diseases.

Ubiquitin carboxyl hydrolase L1 (UCH-L1) is an abundant multifunctional neuron protein. It plays an important role in maintaining the ubiquitin proteasome system (UPS), vital for recognizing and degrading dysfunctional proteins in organisms. In recent decades, UCH-L1 has been implicated in the pathogenesis of many diseases, including neurodegenerative disorders, cancer and diabetes. However, the mechanisms of UCH-L1 involvement have yet to be revealed in detail. Since UCH-L1 contributes many different functions to cell metabolism, its role and regulation might be more complex than previously thought and it has become a research target in many laboratories. In this review, we summarize recent findings related to the actions of UCH-L1 in several human diseases.

An overview on keratinocyte growth factor: from the molecular properties to clinical applications.

KGF (Keratinocyte Growth Factor), also known as FGF7, is a potent mitogen for different types of epithelial cells, which regulates migration and differentiation of these cells and protects them from various insults under stress conditions. KGF is produced by mesenchymal cells and exerts its biological effects via binding to its high-affinity receptor, a splice variant of FGF receptor 2 (FGFR2-IIIb), which is expressed by various types of epithelial cells, including epidermal keratinocytes, intestinal epithelial cells, and hepatocytes. This expression pattern of KGF and its receptor suggests that KGF acts predominantly in a paracrine manner. After acute injury, in various tissues--including the skin, the bladder as well as in chronically injured tissue--KGF expression is strongly up-regulated. This up-regulation is likely to be important for the healing of injured epithelia. In addition, KGF could also exert a protective effect on these cells. There are many researches have been underway to identify clinical applications for KGF. Specifically, KGF is currently being evaluated in clinical trials sponsored by Amgen (Thousand Oaks, CA) to test its ability to ameliorate severe oral mucositis (OM) that results from cancer chemoradiotherapy. In this paper, we provide an overview of the knowledge on molecular properties, biological functions and the recent findings on clinical application of KGF.

Production of polyclonal anti-dUCH (Drosophila ubiquitin carboxyl-terminal hydrolase) antibodies.

Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1), which is a member of the ubiquitin carboxyl-terminal hydrolase (UCH) family, is highly expressed in neurons. In vitro, UCH- L1 exhibits both ubiquitin hydrolase and ligase activity. Many studies have suggested that UCH-L1 is involved in the pathogenesis of Parkinson's disease and some different human cancer diseases, but its role in a living system is still unclear. Recently, Drosophila melanogaster has been shown to be a compatible model for studying human diseases. To investigate the role of UCH-L1 in a living system, the UCH-L1 homologous protein in Drosophila melanogaster (dUCH) is used for analyzing the role of the protein's function in transgenic Drosophila. Here, we used DNA molecular techniques to clone, express, and purify dUCH protein from Escherichia coli. The purified dUCH protein was injected into a rabbit to produce an anti-dUCH antibody, which was shown to have high specificity and sensitivity to the dUCH protein. The affinity of the antibody is 1:320,000 at 7.81 ng/µL antigen concentration. The 1:40,000 dilution-produced antibodies can detect antigen at a low concentration of 0.98 ng/µL. Success in producing this antibody provides good material for further experiments in the study of the role of UCH-L1 by a Drosophila model.

DREF is involved in the steroidogenesis via regulation of shadow gene.

The Drosophila DNA replication-related element-binding factor (dDREF) has been identified as a master regulator of cell proliferation-related genes via its binding to the DRE sequence, 5'-TATCGATA. However, the biological roles of DREF are still to be clarified. Here, we show that DREF mutant females have steroid hormone ecdysone-deficient phenotypes, such as the loss of vitellogenic egg chambers. Furthermore, DREF knockdown in the prothoracic gland of larva prevented pupation and this was rescued via 20-hydroxyecdysone treatment. We found a DRE-like sequence (-625 to -632) in the 5'-flanking region of the Drosophila shadow gene, which catalyzes the conversion of 2-deoxyecdysone to ecdysone, and demonstrated that shadow is a novel target gene of dDREF using quantitative RT-PCR and Chip assays. In addition, we show that the level of dDREF protein correlated with age-related changes in the level of shadow mRNA in the ovaries of wild-type flies. Taken together, our data indicate that dDREF plays a key role in steroid synthesis via regulation of the shadow gene.

Overexpression of ubiquitin carboxyl terminal hydrolase impairs multiple pathways during eye development in Drosophila melanogaster.

UCH-L1 (ubiquitin carboxyl terminal hydrolase L1) is well known as an enzyme that hydrolyzes polyubiquitin at its C-terminal to release ubiquitin monomers. Although the overexpression of UCH-L1 inhibits proteasome activity in cultured cells, its biological significance in living organisms has not been clarified in detail. Here, we utilized Drosophila as a model system to examine the effects of the overexpression of dUCH, a Drosophila homologue of UCH-L1, on development. Overexpression in the eye imaginal discs induced a rough eye phenotype in the adult, at least partly resulting from the induction of caspase-dependent apoptosis followed by compensatory proliferation. Genetic crosses with enhancer trap lines marking the photoreceptor cells also revealed that the overexpression of dUCH specifically impaired R7 photoreceptor cell differentiation with a reduction in activated extracellular-signal-regulated kinase signals. Furthermore, the dUCH-induced rough eye phenotype was rescued by co-expression of the sevenless gene or the Draf gene, a downstream component of the mitogen-activated protein kinase (MAPK) cascade. These results indicate that the overexpression of dUCH impairs R7 photoreceptor cell differentiation by down-regulating the MAPK pathway. Interestingly, this process appears to be independent of its pro-apoptotic function.

Drosophila Myc is required for normal DREF gene expression.

The Drosophila DNA replication-related element-binding factor (dDREF) is required for the expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Finding a canonical E-box, 5'-CACGTG, in the dDREF gene promoter prompted us to explore the possibility that the dDREF gene is a target of Drosophila Myc (dMyc). Luciferase transient expression assays combined with RNA interference in Drosophila S2 cells revealed that knockdown of dmyc reduced dDREF gene promoter activity by 35% to 82%, an effect at least partly mediated by the E-box in the promoter. dm(4)/Y hemizygous mutant larvae demonstrated no maternal dMyc and severe impairment of dDREF mRNA transcription. dMyc loss of function in dm(2)/dm(2) homozygous mutant follicle cell clones also resulted in loss of anti-dDREF immunostaining in nuclei. In contrast, co-expression of dMyc-dMax up-regulated dDREF promoter activity in S2 cells. Furthermore, dMyc over-expressing clones exhibited a high level of dDREF gene expression in wing and eye discs. These results taken together indicate that dMyc is indeed required for dDREF gene expression.