African Monkey Kidney Cells Research Articles

In vitro bioactivity of the fractions and isolated compound from Combretum elaeagnoides leaf extract against selected foodborne pathogens

Ethnopharmacological relevanceCombretum species are used traditionally for the treatment of diarrhoea, hookworm, fever, inflammation, pain and infectious diseases. Infections are commonly caused by the intake of food contaminated with foodborne pathogens. These are a significant concern in the food industry owing to their ability to form biofilms and cause food spoilage, despite the availability of modern food preservation techniques. Combretum elaeagnoides Klotzsch (Combretaceae) is used in southern African traditional medicine against infections and diarrhoea. Aim of the studyThis study evaluated the antimicrobial ability of C. elaeagnoides leaf fractions and the isolated compound quercetin-3-O-rhamnoside against a panel of foodborne pathogens, and biofilms formed by them. The samples were also assessed for their antioxidant activity and cytotoxicity. Materials and methodsFractions prepared from the methanol extract of the leaves, and a bioactive compound (quercetin-3-O-rhamnoside) isolated from the ethyl acetate fraction were investigated for activity against nine reference and clinical strains of foodborne pathogens. The microdilution method was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the fractions and compound. The inhibition of biofilm formation and the crystal violet staining assays were used to determine the antibiofilm efficacy. The DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay and the 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) electron reduction assay were used to determine the antioxidant potential of the fractions and compound. The cytotoxicity was assessed using the 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay against Vero African monkey kidney cells. ResultsThe fractions were active against all tested organisms, with MIC values ranging from 0.03 to 1.25 mg/mL. The best MBC was 0.63 mg/mL. All the fractions and the purified compound inhibited biofilm formation of Staphylococcus aureus and Salmonella Typhimurium, with percentage inhibition values greater than 50% at 1 mg/mL. The compound had very promising antibiofilm activity against Escherichia coli 1 (ATCC 25922) with percentage inhibition of >150%. The compound and fractions had good radical scavenging potential against the DPPH and ABTS radicals. Quercetin-3-O-rhamnoside and the fractions were relatively non-cytotoxic. ConclusionThe ability of the fractions and compound to reduce and inhibit biofilm biomass and their promising antioxidant potential provide motivation to further investigate the use of plants to protect food products from contamination, as well as to treat infections characterized by bacterial biofilms.

Coating with Active Phytomolecules Enhances Anticancer Activity of Bio-Engineered Ag Nanocomplex

Green coating of metal and metal oxide nanomaterials is currently recognized as an eco-friendly route to magnify their biological efficacy and reduce risks due to low biocompatibility. In this study, bio-fabricated metallic silver nanoparticles (AgNPs) were synthesized using three medicinal plant extracts, i.e. Eclipta prostrata, Moringa oleifera and Thespesia populnea and then tested for their cytotoxic activity against human prostate (PC3) and liver (HepG2) cancer cell lines. The green fabricated AgNPs were characterized by Fourier transform infrared spectroscopy, X-ray diffraction, zeta potential analysis, dynamic light scattering, scanning electron microscopy and energy dispersive X-ray spectroscopy. Biofabricated AgNPs exhibited dose-dependent increase in cell toxicity on human prostate cancer, liver cancer and African monkey kidney cell lines. IC50 values of PC3, HepG2 and Vero cells varied depending upon the source used for nanoparticle synthesis. DNA fragmentation, Hoechst, rhodamine and AO/EtBr staining assays confirmed nano-triggered apoptosis of treated cells. The main achievement of this study is that nanofabrication routes relying to E. prostrata, M. oleifera and T. populnea medicinal plant extracts to fabricate Ag nanocomplex within 2 h duration can represent a novel cancer nanodrug and effective way to boost their anticancer efficacy.

The Herpes Simplex Virus Type 1 Glycoprotein D (gD) Cytoplasmic Terminus and Full-Length gE Are Not Essential and Do Not Function in a Redundant Manner for Cytoplasmic Virion Envelopment and Egress

Herpes simplex virus type 1 (HSV-1) acquires its final envelope by budding into cytoplasmic vesicles thought to be derived from trans-Golgi network membranes. This process is facilitated by interactions among the carboxyl termini of viral glycoproteins and tegument proteins. To directly investigate the relative importance of the carboxyl terminus of glycoprotein D (gD) in the presence or absence of gE, a recombinant virus (gDDeltact) was constructed to specify a truncated gD lacking the carboxy-terminal 29 amino acids. Furthermore, two additional recombinant viruses were constructed by mutating from ATG to CTG the initiation codons of gE (gEctg) or both gE and gM (gEctg+gMctg), causing lack of expression of gE or both gE and gM, respectively. A fourth mutant virus was constructed to specify the gEctg+gDDeltact mutations. The replication properties of these viruses were compared to those of a newly constructed recombinant virus unable to express UL20 due to alteration of the two initiation codons of UL20 (UL20ctgctg). All recombinant viruses were constructed by using the double-Red, site-directed mutagenesis system implemented on the HSV-1(F) genome cloned into a bacterial artificial chromosome. The gEctg, gEctg+gMctg, gDDeltact, and gEctg+gDDeltact viruses produced viral plaques on African monkey kidney cells (Vero), as well as other cells, that were on average approximately 30 to 50% smaller than those produced by the wild-type virus HSV-1(F). In contrast, the UL20ctgctg virus produced very small plaques containing three to five cells, as reported previously for the DeltaUL20 virus lacking the entire UL20 gene. Viral replication kinetics of intracellular and extracellular viruses revealed that all recombinant viruses produced viral titers similar to those produced by the wild-type HSV-1(F) virus intracellularly and extracellularly at late times postinfection, with the exception of the UL20ctgctg and DeltaUL20 viruses, which replicated more than two-and-a-half logs less efficiently than HSV-1(F). Electron microscopy confirmed that all viruses, regardless of their different gene mutations, efficiently produced enveloped virions within infected cells, with the exception of the UL20ctgctg and DeltaUL20 viruses, which accumulated high levels of unenveloped virions in the cytoplasm. These results show that the carboxyl terminus of gD and the full-length gE, either alone or in a redundant manner, are not essential in cytoplasmic virion envelopment and egress from infected cells. Similarly, gM and gE do not function alone or in a redundant manner in cytoplasmic envelopment and virion egress, confirming previous findings.

Evaluation of androgen receptor transcriptional activities of some pesticides in vitro

Organophosphates and pyrethroids are among the most common pesticides currently in use worldwide. Several pesticides have been reported to possess hormonal activities, and thus are classified as endocrine disruptors. The present study was planned to evaluate potential androgenic and antiandrogenic activities of the pesticides. The selected chemicals are three organophosphate pesticides including dichlorvos, parathion and trichlorphon, and two pyrethroid pesticides including permethrin and cypermethrin. We evaluated the pesticides for androgen receptor (AR)-mediated mechanisms using a human AR reporter gene assay in African monkey kidney cell line CV-1 transiently transfected with the constructed reporter gene plasmid pMMTV-CAT and the hAR expression plasmid AR/pcDNA3.1. We demonstrated that parathion showed significant inhibitory effects on the transcriptional activity induced by 1 nM of DHT with IC 50 value of (2.01 ± 0.42) × 10 −7 M, though trichlorphon and dichlorvos lacked this activity. The two pyrethroid pesticides permethrin and cypermethrin exhibited lower activity than parathion with IC 50 value of (5.68 ± 2.20) × 10 −5 and (6.80 ± 2.30) × 10 −5 M, respectively. On the other hand, we failed to find AR-mediated androgenic activities of the tested chemicals. It is suggested that parathion possesses the highest activity, and permethrin and cypermethrin acted as poor antiandrogens. The present study provides insight into the mechanism of the pesticides.

Evaluation of androgen receptor transcriptional activities of bisphenol A, octylphenol and nonylphenol in vitro

In this study, we have developed a human androgen receptor (hAR) reporter gene assay using African monkey kidney cell line CV-1 transiently transfected with the constructed reporter gene plasmid pMMTV-CAT and the hAR expression plasmid AR/pcDNA3.1. The assay displayed appropriate response to the known AR agonist 5α-dihydrotestosterone (DHT) and AR antagonist flutamide. DHT induced AR-mediated transcriptional activity in a concentration-dependent manner with median effective concentration (EC 50) value of (3.90 ± 1.43) × 10 −10 M. Flutamide exhibited potent antiandrogenic activity with median inhibitory concentration (IC 50) value of (1.02 ± 0.35) × 10 −7 M. Bisphenol A (BPA) and alkylphenols (APs) belong to the industrial chemicals that have received considerable attention due to high production and widespread usage. We investigated BPA, 4-octylphenol (OP) and 4-nonylphenol (NP) for their agonistic and antagonistic activities by the AR reporter gene assay. BPA showed significant inhibitory effects on the transcriptional activity induced by DHT with IC 50 value of (7.46 ± 1.23) × 10 −7 M. OP and NP exhibited lower antiandrogenic activities than BPA with IC 50 of (9.71 ± 3.82) × 10 −5 M and (2.02 ± 0.90) × 10 −5 M, respectively. On the other hand, we failed to find AR-mediated androgenic activities of the three tested chemicals. In conclusion, BPA, OP and NP all act as AR antagonists, and BPA possesses the highest activity.

Androgen receptor activities of p, p′-DDE, fenvalerate and phoxim detected by androgen receptor reporter gene assay

In this study, we have developed a transient human androgen receptor (hAR) reporter gene assay using African monkey kidney cell line CV-1. The assay displayed appropriate response to the known androgen receptor (AR) agonist 5α-dihydrotestosterone (DHT) and AR antagonist nilutamide. DHT induced AR-mediated transcriptional activity in a concentration-dependent manner with median effective concentration (EC 50) value of 3.90 × 10 −10 M. Nilutamide exhibited potent antiandrogenic activity with median inhibitory concentration (IC 50) value of 8.90 × 10 −7 M. The transcription could not be activated by glucocorticoid receptor (GR) agonist dexamethasone, which suggested that the assay system be highly specific to androgenic compounds without cross-talk to GR agonist. The assay shows acceptable repeatability to DHT with intra coefficient of variability (CV) of 9.3% and inter CV of 10.9%. We applied this assay to evaluate the androgenic and antiandrogenic activities of some pesticides including organochlorine p, p′-dichlorodiphenyldichloroethylene (DDE), pyrethroid fenvalerate and organophosphorus phoxim. The results showed that p, p′-DDE exhibited potent antiandrogenic activity. Fenvalerate acted as weak AR antagonist and phoxim did not show AR antagonistic activity. We failed to find AR agonistic effects of the three pesticides. The present study provided a promising tool to screen for AR agonists and antagonists.

Identification and Functional Characterization of a 5-Transmembrane Domain Variant Isoform of the NTS2 Neurotensin Receptor in Rat Central Nervous System

The present study demonstrated that alternative splicing of the rat nts2 receptor gene generates a 5-transmembrane domain variant isoform (vNTS2) that is co-expressed with the full-length NTS2 receptor throughout the brain and spinal cord, as evidenced by reverse transcription-PCR. The vNTS2 polypeptide is 281 amino acids in length, which is 135 amino acids shorter than the full-length isoform. Immunohistochemical and radioligand binding studies revealed that the HA-tagged recombinant vNTS2 receptor is poorly targeted to plasma membranes in transfected COS-7 cells. Binding studies also showed that the truncated receptor displayed a 5000-fold lower affinity for neurotensin (NT) than its full-length counterpart (IC(50) of 10 mum and 2 nm, respectively). Yet NT binding induced efficient internalization of receptor-ligand complexes in vNTS2-transfected cells. Furthermore, it produced a rapid (<5 min) activation of the mitogen-activated protein kinases (ERK1/2) pathway, indicating functional coupling of the variant receptor. This activation is sustained (>1 h) and is also produced by the NTS2 agonist levocabastine. Western blotting experiments suggested that vNTS2 is not expressed in monomeric form in the rat central nervous system. However, it does appear to form a variety of multimeric complexes, including homodimers and heterodimers, with the full-length NTS2. Indeed, co-immunoprecipitation studies in dually transfected cells demonstrated that the two receptor isoforms can form stable associations. Taken together, the present results indicated that the rat vNTS2 is a functional receptor that may play a role in NT signaling in mammalian central nervous system.

Functional Characterization of the HveA Homolog Specified by African Green Monkey Kidney Cells with a Herpes Simplex Virus Expressing the Green Fluorescence Protein

We cloned the gene specified by African monkey kidney cells (Vero) that codes for the homolog of the herpes virus entry mediator (HveA) specified by HeLa cells. The primary sequence of the monkey HveA (HveAs) differed significantly from HveA. Single amino acid differences were distributed throughout the amino and carboxyl terminal portions of the HveAs in comparison with the HveA, whereas certain regions were highly conserved. The predicted membrane spanning domains of the two receptors differed substantially due to insertions and deletions of short amino acid sequences. The ability of HveAs to mediate HSV virus entry was tested in a series of experiments using the recombinant virus KOS/EGFP, which constitutively expressed the enhanced green fluorescence protein (EGFP) and Chinese hamster ovary cells (CHO) transformed with the HveAs gene. The KOS/EGFP virus was constructed by inserting an EGFP gene cassette within the intergenic region between the UL53 (gK) and UL54 (ICP27) genes. The KOS/EGFP virus formed viral plaques and replicated as well as the wild-type KOS virus. HveAs-transformed CHO cells constitutively expressing HveAs mediated herpesvirus entry efficiently, whereas cells transformed with the HveAs gene in the noncoding orientation did not mediate virus entry. A genetically engineered protein composed of the amino-terminal portion of the HveAs protein fused to the heavy chain of mouse IgG immunoglobulin as well as mouse antibodies raised against HveAs blocked virus entry into HveAs-transformed CHO cells. Thus, HveAs is the functional homolog of HveA.

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A.

The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9-109 ng/ml and between purified CPE at 40-400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A

The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9–109 ng/ml and between purified CPE at 40–400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.

Growth characteristics of two rhinovirus strains in WI-26 and monkey kidney cells.

Haff, R. F. (Smith Kline & French Laboratories, Philadelphia, Pa.), B. Wohlsen, E. E. Force, and R. C. Stewart. Growth characteristics of two rhinovirus strains in WI-26 and monkey kidney cells. J. Bacteriol. 91:2339-2342. 1966.-Viruses with 1059 and HGP serotype and with human and monkey host range characteristics, respectively, were employed. Adsorption kinetics of the 1059 and HGP strains to WI-26 cells, and HGP to Green African monkey kidney cells (MKC), were similar. Fifty per cent of the virus was adsorbed to cell monolayers within 10 min; adsorption was essentially complete by 2 hr. The 1059 strain failed to adsorb to MKC, at least to an appreciable extent. Lack of receptors for adsorption of 1059 accounts for the inability of this cell to support multiplication of the virus. It is probable that MKC are refractory to infection with other H strains of rhinovirus for the same reason. Single-step multiplication cycles have been described for the HGP strain in WI-26 and MKC cultures and for the 1059 strain in WI-26 cells. In both cells, HGP exhibited a latent period of 7 hr. Increase of intracellular and cell-associated virus appeared somewhat prior to that of extracellular virus. Maximal titers were attained by 9 to 10 hr. In contrast, initial increase of 1059 in WI-26 cells occurred after 10 hr. Titer rose to peak level 15 hr after infection. Yield of 1059 in WI-26 cells was also fivefold lower than that of HGP in either cell system.