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.
Toxoplasma gondii is the causative agent for toxoplasmosis. The rhoptry protein 1 (ROP1) is secreted by rhoptry, an apical secretory organelle of the parasite. ROP1 plays an important role in host cell invasion. In this study, the efficacy of ROP1 as a vaccine candidate against toxoplasmosis was evaluated through intramuscular or subcutaneous injection of BALB/c mice followed by immunological characterization (humoral- and cellular-mediated) and lethal challenge against virulent T. gondii RH strain in BALB/c mice. Briefly, a recombinant DNA plasmid (pVAX1-GFP-ROP1) was expressed in CHO cells while expression of recombinant ROP1 protein (rROP1) was carried out in Escherichia coli expression system. Immunization study involved injection of the recombinant pVAX1-ROP1 and purified rROP1 into different group of mice. Empty vector and PBS served as two different types of negative controls. Results obtained demonstrated that ROP1 is an immunogenic antigen that induced humoral immune response whereby detection of a protein band with expected size of 43 kDa was observed against vaccinated mice sera through western blot analysis. ROP1 antigen was shown to elicit cellular-mediated immunity as well whereby stimulated splenocytes with total lysate antigen (TLA) and rROP1 from pVAX1-ROP1 and rROP1-immunized mice, respectively, readily proliferated and secreted large amount of IFN-γ (712 ± 28.1 pg/ml and 1457 ± 31.19 pg/ml, respectively) and relatively low IL-4 level (94 ± 14.5 pg/ml and 186 ± 14.17 pg/ml, respectively). These phenomena suggested that Th1-favored immunity was being induced. Vaccination with ROP1 antigen was able to provide partial protection in the vaccinated mice against lethal challenge with virulent RH strain of tachyzoites. These findings proposed that the ROP1 antigen is a potential candidate for the development of vaccine against toxoplasmosis.
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.
The use of herbal formulations has gained scientific interest, particularly in cancer treatment. In this study, the herbal formulation of interest, denoted as C168, is a mixture of eight genera of plants. This study aims to investigate the antiproliferative effect of C168 methanol extract (CME) on various cancer cells and its underlying mechanism of action on the most responsive cell line, namely, HCT 116 cells. CME exerted antiproliferative activities on HCT 116 colorectal carcinoma cells and HepG2 hepatocellular carcinoma cells but not on CCD-841-CoN normal colon epithelial cells, Jurkat E6.1 lymphoblastic leukemic cells, and V79-4 Chinese hamster lung fibroblasts. Further investigation on HCT 116 cells showed that CME induced G2/M cell-cycle arrest and apoptosis. Treatment of CME induced oxidative stress in HCT 116 cells by increasing the superoxide anion level and decreasing the intracellular glutathione. CME also increased tail moment value and H2AX phosphorylation in HCT 116 cells, suggesting DNA damage as an early signal of CME induced apoptosis. Loss of mitochondrial membrane potential in CME-treated cells also indicated the involvement of mitochondria in CME induced apoptosis. This study indicated the selectivity of CME toward colon cancer cells with the involvement of oxidative damage as its possible mechanism of action.
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.
Nipah virus (NiV) is a recently emerged zoonotic Paramyxovirus that causes regular outbreaks in East Asia with mortality rate exceeding 75%. Major cellular targets of NiV infection are endothelial cells and neurons. To better understand virus-host interaction, we analyzed the transcriptome profile of NiV infection in primary human umbilical vein endothelial cells. We further assessed some of the obtained results by in vitro and in vivo methods in a hamster model and in brain samples from NiV-infected patients. We found that NiV infection strongly induces genes involved in interferon response in endothelial cells. Among the top ten upregulated genes, we identified the chemokine CXCL10 (interferon-induced protein 10, IP-10), an important chemoattractant involved in the generation of inflammatory immune response and neurotoxicity. In NiV-infected hamsters, which develop pathology similar to what is seen in humans, expression of CXCL10 mRNA was induced in different organs with kinetics that followed NiV replication. Finally, we showed intense staining for CXCL10 in the brain of patients who succumbed to lethal NiV infection during the outbreak in Malaysia, confirming induction of this chemokine in fatal human infections. This study sheds new light on NiV pathogenesis, indicating the role of CXCL10 during the course of infection and suggests that this chemokine may serve as a potential new marker for lethal NiV encephalitis.
The mosquito-borne chikungunya virus (CHIKV) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. Although there are several studies to evaluate the efficacy of drugs against CHIKV, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. Here, we investigated the antiviral activity of three types of flavonoids against CHIKV in vitro replication. Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Antiviral activity of effective compound was further investigated by evaluation of CHIKV protein expression using western blotting for CHIKV nsP1, nsP3, and E2E1 proteins. Briefly, silymarin exhibited significant antiviral activity against CHIKV, reducing both CHIKV replication efficiency and down-regulating production of viral proteins involved in replication. This study may have important consequence for broaden the chance of getting the effective antiviral for CHIKV infection.
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.