Monoamine oxidases (MAOs) and muscarinic acetylcholine receptors (mAChRs) are considered important therapeutic targets for Parkinson's disease (PD). Lipophilic tanshinones are major phytoconstituents in the dried roots of Salvia miltiorrhiza that have demonstrated neuroprotective effects against dopaminergic neurotoxins and the inhibition of MAO-A. Since MAO-B inhibition is considered an effective therapeutic strategy for PD, we tested the inhibitory activities of three abundant tanshinone congeners against recombinant human MAO (hMAO) isoenzymes through in vitro experiments. In our study, tanshinone I (1) exhibited the highest potency against hMAO-A, followed by tanshinone IIA and cryptotanshinone, with an IC50 less than 10 µM. They also suppressed hMAO-B activity, with an IC50 below 25 µM. Although tanshinones are known to inhibit hMAO-A, their enzyme inhibition mechanism and binding sites have yet to be investigated. Enzyme kinetics and molecular docking studies have revealed the mode of inhibition and interactions of tanshinones during enzyme inhibition. Proteochemometric modeling predicted mAChRs as possible pharmacological targets of 1, and in vitro functional assays confirmed the selective M4 antagonist nature of 1 (56.1% ± 2.40% inhibition of control agonist response at 100 µM). These findings indicate that 1 is a potential therapeutic molecule for managing the motor dysfunction and depression associated with PD.
Generation of high-producing clones is a perquisite for achieving recombinant protein yields suitable for biopharmaceutical production. However, in many industrially important cell lines used to produce recombinant proteins such as Chinese hamster ovary, mouse myeloma line (NS0), and hybridomas, only a minority of clones show significantly above-average productivity. Thus, in order to have a reasonable probability of finding rare high-producing clones, a large number of clones need to be screened. Limiting dilution cloning is the most commonly used method, owing to its relative simplicity and low cost. However the use of liquid media in this method makes the selection of monoclonal hybridoma and transfectoma colonies to be labor intensive and time consuming, thus significantly limiting the number of clones that can be feasibly screened. Hence, we describe the use of semisolid media to immobilize clones and a high-throughput, automated colony picker (ClonePix FL) to efficiently isolate monoclonal high-producing clones secreting monoclonal antibodies.
According to the World Cancer Research Fund International (WCRFI), breast cancer is the most common type of cancer in women worldwide with recorded 1.7 million new cases in 2012. The main line of treatments is still limited to chemotherapy, surgery and radiotherapy which could lead to a wide range of dangerous side effects. This study was conducted to evaluate the effect of low intensity ultrasound (LIUS) on cell proliferation, percentage of living and dead cells and the induction of apoptosis on the MCF-7 cell line with CHO cells as the control for non-cancerous group. In order to achieve the objective of this study, several methods of cell-bioguided assays were used including the MTT assay for cell proliferation, Live/Dead assay for the determination of both live and dead cells and gene expression study for the detection of apoptosis in the cells. The cytotoxicity and Live/Dead assays data provided preliminary data that the LIUS has potential to induce apoptosis in a wide population of breast cancer cells. Furthermore, the LIUS treatment induced the expression of p53-mRNA at a detectable level via qPCR analysis, indicating the activation of apoptosis. In short, our study suggested LIUS dosage used in this study could potentially show positive effects in the induction of apoptosis selectively on the MCF-7 with less harm to the control CHO cells.
Goniothalamin (GTN) is a styrylpyrrone derivative from Goniothalamus umbrosus and other Annonaceae species. It has been shown to have anti-cancer and apoptosis-inducing properties against various human tumour and animal cell lines. The compound has also been shown to be active in vivo against DMBA-induced rat mammary tumours and was reported as an anti-fertility agent in rats. The aim of our study was to assess the genotoxicity of GTN in CHO cells using the UKEMS guidelines. A metabolic activation fraction (S9) was prepared according to standard methods. The methylthiazoletetrazolium (MTT) screening assay was then carried out to determine the cytotoxicity index (IC50) of GTN. The average IC50 value was 12.45 (+/- 3.63)microM. The mitotic index (MI) assay was then performed to determine the clastogenicity indices (MI(C25), MI(C50) and MI(C100)) of GTN. The chromosome aberration (CA) induction assay using air-dried metaphase spread was then performed to investigate the clastogenic effects of goniothalamin. Benzo[a]pyrene (BaP) and ethylmethanesulphonate (EMS) were used as positive controls in the presence and absence of S9 metabolic activation, respectively. The anti-genotoxicity effect of GTN was also assessed using a combination of GTN and EMS, and GTN and BaP. Dose-responses of CA frequencies were determined for both, the genotoxicity and anti-genotoxicity effects. GTN on its own and when combined with positive controls, was found to induce and enhance CA, respectively. Chromatid and whole chromosome breaks/gaps, as well as interchanges, endoreduplications and ring chromosomes were the main types of aberration induced by GTN. The overall clastogenic effect of GTN was statistically significant. In conclusion, GTN is potentially a genotoxic or clastogenic substance without any anti-genotoxic properties.
The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus.
Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.
Serum deprivation inhibits cell growth and initiates apoptosis cell death in mammalian cell cultures. Since apoptosis is a genetically controlled cell death pathway, over-expression of anti-apoptotic proteins may provide a way to delay apoptosis. This study investigated the ability of the X-linked inhibitor of apoptosis protein (XIAP) to inhibit apoptosis induced by serum deprivation. Study includes evaluation of the ability of XIAP to prolong culture period and its effect on cell proliferation in serum-deprived media. The full length human XIAP was introduced into CHO-K1 cell lines and the effects of XIAP over-expression on the inhibition of apoptosis induced by serum-deprived conditions were examined. In batch cultures, cells over-expressing XIAP showed decreased levels of apoptosis and a higher number of viable cell under serum-deprived conditions compared to the control cell lines. The viability of control cells dropped to 40% after 2days of serum deprivation, the XIAP expressing cells still maintained at a viability higher than 90%. Further investigation revealed that the caspase-3 activity of the CHO-K1 cell line was inhibited as a result of XIAP expression.
The effect of mild hypothermic (32 degrees C) conditions on cell growth, cell-cycle progress, and antibody production of hybridoma C2E7 cells was investigated in the present study. The growth of hybridoma cells was slower during the mild hypothermic condition compared to that at 37 degrees C; this led to about 10% decrease in maximum viable cell density and volumetric antibody productivity. However, under mild hypothermic growth conditions, the culture viability was substantially improved and the specific antibody productivity was enhanced compared to that at 37 degrees C. The average specific productivity for the entire batch culture at 32 degrees C is about 5% higher than that at 37 degrees C. Cell-cycle analysis data showed that there was no growth arrestment during the mild hypothermic growth of hybridoma cells. The G1-phase cells were increased, while the S-phase cells were decreased gradually as the culture time progressed. Further analysis showed that the specific antibody productivity of hybridoma cells was correlated to the fraction of S-phase cells.
Selected indole-based kratom alkaloids were evaluated for their opioid and adrenergic receptor binding and functional effects, in vivo antinociceptive effects, plasma protein binding, and metabolic stability. Mitragynine, the major alkaloid in Mitragyna speciosa (kratom), had higher affinity at opioid receptors than at adrenergic receptors while the vice versa was observed for corynantheidine. The observed polypharmacology of kratom alkaloids may support its utilization to treat opioid use disorder and withdrawal.
Lignosus rhinocerotis mushroom is widely used as traditional medicine and as soup ingredient in Malaysia and Hong Kong. Its sclerotium is the part of edibility and is traditionally used for the treatment of fever, cough, asthma and cancer. In view of its safety profile, very little information is found in scientific literature.
Andrographolide, a major bioactive compound isolated from Andrographis paniculata (Burm. F.) Nees, was evaluated for its effects on the hOAT1 membrane transporter. Substrate determination and inhibition of hOAT1-mediated uptake transport assay was carried out using recombinant CHO-hOAT1 cells. The results showed that the uptake ratio of andrographolide was less than 2.0 at all concentrations tested, indicating that andrographolide is not a hOAT1 substrate. Andrographolide has no significant effects on the p-aminohippuric acid uptake and on the mRNA and protein expression of hOAT1. In conclusion, andrographolide may not pose a drug-herb interaction risk related to hOAT1.
G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha(2A) adrenergic receptor (α(2A)-R) by the ubiquitous stress-inducible 70kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α(2A)-R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α(2A)-R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α(2A)-R-catalyzed [(35)S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine(1A) receptor (5-HT(1A)-R). In heat-stressed CHO cells, the α(2A)-R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α(2A)-R compared to the 5-HT(1A)-R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.
Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377-588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.
Zerumbone (ZER), a potential anticancer compound, isolated from the fresh rhizomes of Zingiber zerumbet. In this investigation, the cytotoxic properties of ZER were evaluated, on cancer cells of human cervix (HeLa), breast and ovary, and normal cells of Chinese Hamster ovary, using MTT assay. Apoptogenic effects of ZER on HeLa were studied using fluorescence microscopy (AO/PI double staining), scanning and transmission electron microscopy (SEM and TEM), and colorimetric assay of the apoptosis promoter enzyme, caspase-3. The results of MTT assay showed that ZER has less effect on normal cells compared to cancer cells. The lowest IC(50) of ZER was observed on HeLa cells. Cytological observations showed nuclear and chromatin condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, holes, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by double staining of AO/PI, SEM and TEM. Statistical analysis (two-tailed t-test) of differential counting of 200 cells under fluorescence microscope revealed significant difference in apoptotic cells populations between treated and untreated HeLa cells. In addition, ZER has increased the cellular level of caspase-3 on the treated HeLa cells. It could be concluded that ZER was able to produce distinctive morphological features of cell death that corresponds to apoptosis.
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with epithelial-cell cancers and B cell lymphomas. An effective EBV vaccine is not available. We found that antibodies to the EBV glycoprotein gH/gL complex were the principal components in human plasma that neutralized infection of epithelial cells and that antibodies to gH/gL and gp42 contributed to B cell neutralization. Immunization of mice and nonhuman primates with nanoparticle vaccines that displayed components of the viral-fusion machinery EBV gH/gL or gH/gL/gp42 elicited antibodies that potently neutralized both epithelial-cell and B cell infection. Immune serum from nonhuman primates inhibited EBV-glycoprotein-mediated fusion of epithelial cells and B cells and targeted an epitope critical for virus-cell fusion. Therefore, unlike the leading EBV gp350 vaccine candidate, which only protects B cells from infection, these EBV nanoparticle vaccines elicit antibodies that inhibit the virus-fusion apparatus and provide cell-type-independent protection from virus infection.
AbstractToxoplasma gondii infects a broad range of warm-blooded hosts, including humans. Important clinical manifestations include encephalitis in immunocompromised patients as well as miscarriage and fetal damage during early pregnancy. Toxoplasma gondii dense granule antigen 2 and 5 (GRA2 and GRA5) are essential for parasitophorous vacuole development of the parasite. To evaluate the potential of GRA2 and GRA5 as recombinant DNA vaccine candidates, these antigens were cloned into eukaryotic expression vector (pcDNA 3.1C) and evaluated in vaccination experiments. Recombinant DNA vaccines constructed with genes encoding GRAs were validated in Chinese hamster ovary cells before evaluation using lethal challenge of the virulent T. gondii RH strain in BALB/c mice. The DNA vaccines of pcGRA2 and pcGRA5 elicited cellular-mediated immune response with significantly higher levels of interferon-gamma, interleukin-2 (IL-2), IL-4, and IL-10 (P < 0.05) compared with controls. A mixed T-helper cell 1 (Th1)/Th2 response was associated with slightly prolonged survival. These findings provide evidence that DNA vaccination with GRA2 and GRA5 is associated with Th1-like cell-mediated immune responses. It will be worthwhile to construct recombinant multiantigen combining full-length GRA2 or/and GRA5 with various antigenic proteins such as the surface antigens and rhoptry antigens to improve vaccination efficacy.
Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors' ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3-6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 μM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.
Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM) models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP) scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7-11.2 μM) as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5-40.2 μM). Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 μM), hepatotoxicity (up to 30 μM) or cardiotoxicity (up to 30 μM).