Syrian Hamster Kidney Research Articles

In English

Today, the antiviral activity of oxide nanomaterials can be used in the fight against the viral disease COVID-19. It is thought that Ag nanoparticles may bind to the surface glycoprotein of the virus and interfere with the virus’s interaction with epithelial cells, and inhibit virus reproduction by releasing silver ions in the cell. The viruses’ inhibition with RNA (ribonucleic acid) and DNA (deoxyribonucleic acid) genomes by oxide nanocomposites action was presented. In this research, the surface structure of doped CeO2 (La2O3) was studied by nitrogen adsorption-desorption based on BET method. The silver atom’s existence in CeO2 - Ag0 can facilitate the transport of more holes to the surface and can enhance the optical, antivirus activity. The primary particle size of pure cerium dioxide is 7 nm, for CeO2 - Ag composite at 2 and 4 wt. % of silver is 6.5 and 6.9 nm; for La2O3 - Ag 27 and 35 nm, respectively. Cell viability was assessed using an MTT (3-(4,5-Dimethylthiazol 2-yl)-2,5-diphenyltetrazolium bromide) assay after NPs (nanoparticles) exposure, since only viable cells have functional mitochondrial dehydrogenase enzymes that can reduce MTT to formazan. Nanoparticles were non-toxic for BHK-21(Syrian hamster kidney), Hep-2 (Human larynx carcinoma), and MDCK (Canine kidney) cells in concentrations of 10 and 100 μg/ml, while cell viability was within 76÷100 %. La2O3 and CeO2, which contained 4 wt. % of Ag at a concentration of 1000 μg/ml had a lower toxic effect: for BHK-21 cells 68 and 76 % of viable cells, respectively; for Hep-2 - 40 and 36 %, for MDCK - 42 and 48 %; La2O3 and CeO2 with 2 and 5 wt. % of Ag at a concentration of 1000 µg/ml were highly toxic. The level of ВНК-21, Нер-2, and MDCK cells viability was in a range of 7 to 37 %. It has been stated that oxides of cerium and lanthanum have a pronounced virucidal action against the Herpes simplex virus and Influenza A virus by completely inhibiting the development of its cytopathic action. The lanthanum and cerium oxides with 2 and 5 wt. % of silver inhibited the development of the virus’s CPE by more than 5.0 log10 compared to the virus control. The results show that lanthanum and cerium oxides with 2 and 5 wt. % silver have a high virucidal effect against herpes simplex virus type 1. A 1.0÷4.0 log10 reduction in the infectious titer of the Herpes virus synthesized “de novo” in the presence of lanthanum and cerium oxide nanocomposites has been shown.

CHARACTERISTICS OF THE EQUINE RHINOPNEUMONIA VIRUS AK-2011

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks, and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0 lg TCID50/cm3. Sequencing and polymerase chain reaction (PCR) analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPE) induced by the AK-2011 virus stain (dilution 101) in calf trachea and horse kidney cell cultures were stable from the 1st to 10th passages, with biological activity of 5.75-6.00 lg TCID50/cm3. CPE caused by the virus were apparent on days 2-3, further developed intensively, and extended to 60-80% of the cell monolayer on days 5-7. In calf kidney, sheep kidney, green monkey kidney, and bovine kidney cell cultures, the same changes were observed 1-2 days later. The changes were slow, and by day 7-10 CPE extended to no more than 30-50% of ​​the cell monolayer. An attenuated strain AK-2011 of equine rhinopneumonitis virus was obtained. It was considered a candidate for the manufacture of a vaccine for equine rhinopneumonitis. Thus, the attenuated strain AK-2011 of the equine rhinopneumonitis virus was characterized.

Real-World Study on Chai-Shi-Jie-Du Granules for the Treatment of Dengue Fever and the Possible Mechanisms Based on Network Pharmacology.

Traditional Chinese medicine (TCM) is a widely used method for treating dengue fever in China. TCM improves the symptoms of patients with dengue, but there is no standard TCM prescription for dengue fever. This real-world study aimed to evaluate the effects of Chai-Shi-Jie-Du (CSJD) granules for the treatment of dengue fever and the underlying mechanisms. We implemented a multicenter real-world study, an in vitro assay and network pharmacology analysis. Patients from 5 hospitals in mainland China who received supportive western treatment in the absence or presence of CSJD were assigned to the control and CSJD groups between 1 August and 31 December 2019. Propensity score matching (PSM) was performed to correct for biases between groups. The clinical data were compared and analyzed. The antidengue virus activity of CSJD was tested in Syrian baby hamster kidney (BHK) cells using the DENV2-NGC strain. Network pharmacological approaches along with active compound screening, target prediction, and GO and KEGG enrichment analyses were used to explore the underlying molecular mechanisms. 137 pairs of patients were successfully matched according to age, sex, and the time from onset to presentation. The time to defervescence (1.7 days vs. 2.5 days, P < 0.05) and the disease course (4.1 days vs. 6.1 days, P < 0.05) were significantly shorter in the CSJD group than those in the control group. CSJD showed no anti-DENV2-NGC virus activity in BHK cells. Network pharmacology analysis revealed 108 potential therapeutic targets, and the top GO and KEGG terms were related to immunity, oxidative stress response, and the response to lipopolysaccharide. CSJD granules exhibit high potential for the treatment of dengue fever, and the therapeutic mechanisms involved could be related to regulating immunity, moderating the oxidative stress response, and the response to lipopolysaccharide.

AK-2011 strain for the development of a vaccine against equine rhinopneumonitis.

Equine rhinopneumonitis is an acute, highly contagious disease found virtually worldwide. The purpose of the studies presented in this paper is to develop a technology for the manufacture of a cell-derived equine rhinopneumonitis vaccine, as well as to assess the safety and immunogenicity of the newly developed vaccine in laboratory animals model. The object of the studies was the AK-2011 strain isolated from the horses suffering from rhinopneumonitis during an outbreak of abortions. The viability of the AK-2011 strain was assessed using a continuous line of calf trachea cells, a continuous line of calf kidney cells, a continuous line of sheep kidney cells, a continuous line of bovine kidney cells, a continuous line of green monkey kidney cells, a continuous line of Syrian hamster kidney cells, a primary trypsinized culture of horse kidney cells grown in tubes and flasks and the AK-2011 laboratory strain of equine rhinopneumonitis virus with biological activity of 6.0lg TCID50/cm 3 . Sequencing and polymerase chain reaction analysis were performed. The virus isolated from the ORF68 gene in Kazakhstan appeared to be the most similar to the T-953 and 2222-03 strains isolated in the USA and Australia, respectively, in terms of phylogenetics. As to primary infections, cytopathic effects (CPEs) induced by the AK-2011 virus stain (dilution 101 ) in calf trachea and horse kidney cell cultures were stable from the first to tenth passages, with biological activity of 5.75-6.00 lg TCID50/cm 3 . CPEs caused by the virus were apparent on days 2-3, further developed intensively and extended to 60-80% of the cell monolayer on days 5-7. The vaccine results can be used to immunize horses on farms against rhinopneumonia, and horses should be immunized twice with an interval of 2-3 months.

Inactivated vaccine against Aujeszky's disease.

Background and Aim:The Aujeszky’s disease, also known as Pseudorabies, remains one of the most problematic fulminant diseases in domestic animals, affecting the central nervous system. The study aimed to investigate the effect of an inactivated vaccine against Aujeszky’s disease based on “Kordai” virus strain.Materials and Methods:To test the inactivation of the “Kordai” strain (grown by the roller method in VNK-21/13 cell culture with an infectious titer of at least 7.5 lg TCD50/ml) which is causative of Aujeszky’s disease, next-generation teotropin and propolis preparations were usedin concentrations of 0.1%, 0.08%, and 0.04%.Results:As a result of comparative studies on the optimization of parameters for inactivating the “Kordai” virus strain, it was established that teotropin is a more effective inactivant than propolis. At the same time, the optimal final concentration of teotropin for inactivation was 0.1%, along with a reaction medium temperature of 37°C, pH of 7.4-7.6, and duration of inactivation of 14 h. The titer of virus-neutralizing activity (VNA) of antibodies at the pH (neutralization reactions) in vaccinated sheep of 10-12 months of age was 7.5±0.3, Ig TCID50/ml (tissue culture infectious dose 50%), and 3.5±0.3 in the cell culture VNK-21/13 (culture of Syrian hamster kidney cells).Conclusion:To determine colostral immunity in newborn lambs, the method of metabolic status correction was used to vaccinate lambs obtained from immune sheep 4 months after birth. The results showed that lambs obtained from immune sheep had high VNA titers. A sustained immune response in vaccinated animals was obtained after double vaccination.

Studies of biological properties of continuous suspension ВНК-21/SUSP/ARRIAH cell line

The results of the studies of cytomorphological, karyological, cultural properties of continuous suspension ВНК-21/SUSP/ARRIAH subline of newborn Syrian hamster kidney cells intended for foot-and-mouth disease, rabies, bovine parainfluenza-3, Aujeszky’s disease virus reproduction, as well as for production of diagnostic veterinary biologicals are presented. When cultured in suspension, BHK-21/SUSP/ARRIAH cell subline undergoes selection towards hypoploidy: modal class is represented by cells with 41 chromosomes (32–40% of cells); the share of cells containing 40–42 chromosomes is 78–80%; the share of polyploids averages around 1%; the range of variation in the number of chromosomes is from 36 to 54. BHK-21/SUSP/ARRIAH cell subline cultured in suspension with cell seeding concentration of 0.6–0.8 million cells/cm3 demonstrates growth rate of 6.67–11.00 and 96–99% cell viability. After 48 hours, G1-phase (diploid-2n) cells prevail in the cell population of the new subline (30.0–75.0% of cells); cells that undergo preparation for mitosis (S-phase) and mitosis (G2+M-phase) account for 3.0 to 20.0% of the entire population; the number of meganucleated and multinucleated cells (&gt;4n) at the beginning and at the end of the logarithmic phase increases to 2%. BHK-21/SUSP/ARRIAH cells recover rapidly after cryopreservation and demonstrate 95–99% viability and growth rate of 3.36–5.88 at passages 1 to 3 and 6.85–10.95 at passages 4 to 12. Continuous suspension BHK-21/SUSP/ARRIAH cell line ensures virus accumulation at the following titres: FMD virus – 7.30– 8.00 lg TCID50/cm3, rabies virus – 7.25–8.00 lg CCID50/cm3, bovine parainflunza-3 virus – at least 6.00 lg TCID50/cm3, Aujeszky’s disease virus – 7.50–7.80 lg TCID50/cm3.

ДИНАМИКА АДАПТАЦИИ ВИРУСА БЕШЕНСТВА ВАКЦИННОГО ШТАММА «RV-97» К МОНОСЛОЙНОЙ КУЛЬТУРЕ КЛЕТОК ВНК-21/13

The problem of rabies as one of the most dangerous zoonoses continues to be relevant almost all over the world. In development of a live vaccine, an important stage is to obtain an active component - a virus that retains the given phenotypic properties, pathogen cultivation system plays the main role. The aim of this work was to adapt the rabies virus of “RV-97” strain to the finite cell line of the Syrian hamster kidney (VNK -21/13) of the Swedish subline, as well as to carry out a comparative analysis of virus accumulation at different passages. The number of passages that need to be carried out for adaptation of RV-97 strain to the monolayer culture of VNK -21/13 cells was determined. We used a 2-day culture of VNK -21/13 cells in the phase of logarithmic growth (80-90% formation of a cell monolayer). VNK -21/13 cell culture grown as a monolayer in the wells of flat-bottomed plastic plates was used as a test system for infectious activity. A fluorescent label was used to indicate infected cells. It was determined that the smallest number of passages at which the rabies virus of “RV-97” strain is adapted to the finite cell culture of VNK -21/13 of the Swedish subline is the 6th passage level. It was found that the titer of infectious activity of attenuated rabies virus of “RV-97” strain at the 6th passage level is 7.33 ± 0.17 lg CCID50 / cm3.

Санація перещеплюваних ліній клітин від мікоплазменної інфекції

From the early 50's of last century and to nowadays the cell culture lines are a cornerstone in virological, cytological, biochemical and other biological studies. Cell cultures are essential in studying of cellular regulation mechanisms, virus interaction processes during their replication in sensitive cells, and in production of biologically active materials, including vaccines, enzymes, hormones, and monoclonal antibodies. However, today there is very common and often catastrophic problem that connected with cell line contamination by other microorganisms. Contamination with mycoplasma is of particular concern to scientists around the world. Due to difficulties in its detection, it stays unnoticed in cell cultures, having detrimental effect on cell function and morphological status. Cell cultures contamination with mycoplasmas can lead to degenerative changes in cells and their complete loss and represents a potential source of artifacts in cytological, virological and biochemical studies. Mycoplasma-contaminated cell lines are a major problem in research center laboratories and biotechnology facilities. Mycoplasmas are ultramicroscopic free-living prokaryotes whose sizes range from 200 to 400 nm. They lack a cell wall, which makes it impossible to detect them even with an ordinary microscope. In addition, mycoplasmas do not cause turbidity in cell culture media, which often accompanies bacterial or fungal cell culture contamination. The most important thing is that mycoplasma infection usually does not lead to cell death. Therefore, they can multiply and go unnoticed in a cell culture flask for a long period, becoming a major obstacle to reliable and standard in vitro experiments. Nowadays, the rehabilitation of the cell line from mycoplasmas is a very complex and urgent problem. According to many researchers, their attempts to decontaminate cell culture from mycoplasmas have been ineffective. Due to conclusions from their work, all cell lines infected with mycoplasmas are subject to immediate destruction. At the same time, the purchase of new cell lines requires considerable material costs. Therefore, there is a need to find real approaches in the rehabilitation of cell culture lines in research and production laboratories. The article represents the results of experimental studies focus on the decontamination of 12 cultures cell lines of animal origin from mycoplasmas. The research was conducted during 2019-2020 at the department of biotechnology and viral vaccine control (culture line sector) of State scientific control institute of biotechnology and strains of microorganisms. The cell culture lines with PCR-detected mycoplasma infection were used in the experiments, namely: FLK - sheep embryonic kidney cell line; PO - sheep kidney cell line; RK-13- rabbit kidney cell line; RK-15 - pig kidney cell line; MDBK- bovine kidney embryo cells; Vero- African green monkey kidney cells; Marc-145 - African green monkey kidney-derived MA-104 cells; BGM - African green monkey kidney cells; BHK-21 / clone 13 - Syrian hamster kidney cells; KST (KST) - cells of coronary vessels of cattle; SPEV (original name - SNEV) - pig kidney cells; A-72 - culture of subcutaneous tumor cells in dogs. On the early stages of the study, the optimal allowable concentration of the drug ciprofloxacin, which was used in our laboratory to rehabilitate cell cultures from mycoplasmas, was determined. It has been experimentally established that ciprofloxacin at concentration of 20 μg/cm3 completely inhibits the reproduction and life cycle of mycoplasmas and does not cause a significant effect on the cells themselves. Ciprofloxacin in concentration of 20 μg/cm3 was added into the growth and maintenance medium for five consecutive passages to the flasks with mycoplasma-contaminated cell cultures. After forming a cell monolayer in the flasks, it was always thoroughly washed five times with Hanks' solution, and only then the medium was replaced with a supportive one. Thus, the full cycle of cell culture remediation, from the moment of seeding the cells in the flasks until the next reseeding of cells took 3-4 days. 5 cycles were performed with each cell culture affected by mycoplasmas. At the completion of rehabilitation process, the samples of cell culture supernatant from each cell culture line were collected and examined by PCR for the presence of mycoplasmas. As a result, after remediation process of cell cultures mycoplasma infection was detected in only one sample. The efficiency is 88.9% and it proves that the drug ciprofloxacin can be successfully used for decontamination of cell cultures in scientific and industrial virological laboratories.

Detection of Cell Viability via Fluorescence Labeling of Silicate Phosphor with a Low-Temperature Superlong Persistent Luminescence

Cell viability detection is usually accompanied by large errors and toxicity. For the accuracy and safety of detection, it is urgent to develop a practical approach to realize the real-time monitoring of cell viability. Here, a low-temperature persistent phosphor was employed to facilitate the detection of cell viability. Stable low-temperature persistent luminescence originating from Ba4Si6O16:Eu2+, Pr3+ (BSEP) was developed, which could be quantified to describe the viability of baby Syrian hamster kidney (BHK-21) cells. The defect state structure guarantees the excellent persistent luminescence at 193 K of more than 200 h of the as-prepared BSEP phosphor, which ensures the long-term biological application. The structural thermal stability and water tolerance of BSEP suggest the feasibility of the practical application. Our results provide the underlying insights of the low-temperature persistent phosphors for real-time biological detection.

Mutation of I176R in the E coding region weakens Japanese encephalitis virus neurovirulence, but not its growth rate in BHK-21 cells.

Previously, we isolated the Japanese encephalitis virus (JEV) strain SCYA201201. In this study, we passed the SCYA201201 strain in Syrian baby hamster kidney (BHK-21) cells 120 times to obtain the SCYA201201-0901 strain, which exhibited an attenuated phenotype in mice. Comparison of SCYA201201-0901 amino acid sequences with those of other JEV strains revealed a single mutation, I176R, in the E coding region. Using reverse genetic technology, we provide evidence that this single E-I176R mutation does not affect virus growth in BHK-21 cells but significantly decreases JEV neurovirulence in mice. This study provides critical information for understanding the molecular mechanism of JEV attenuation.

Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

MOLECULAR AND BIOLOGICAL CHARACTERISTICS, AND CULTIVATION OF AN ATTENUATED STRAIN 1974-VNIIVViM OF RIFT VALLEY FEVER VIRUS

In recent decades, a threat of spreading Rift Valley fever (RVF) to Asia and Europe raises serious concerns. Within many years, vaccines against RVF were developed in several ways including designing attenuated vaccine versions, inactivated or genetically engineered vaccines. A serious hazard of the RVF agent requires developing sufficiently immunogenic preparations providing both RVF specific prevention and safety precautions in production procedures. Therefore, this work was aimed at molecular and biological characterization of an attenuated RVF virus (RVFV) strain 1974VNIIVViM including search for high-tech cell culture systems for the virus growth and determination of its antigenic and/or immunogenic relationship with a virulent strain Entebbe. The results of the investigations showed that the virus accumulated in continuous cell cultures like saiga kidney (SK), Siberian mountain goat kidney (PSGK-60) and MDVK (calve kidney cell culture) at high titers with infectious (8.53±0.21 lg MICLD50/cm) and antigenic (1:64 to 1:128 in the passive hemagglutination test, PHT) activity levels. We determined optimal regime of the virus growth in BHK-21/13 (Syrian hamster kidney cell culture) using a roller culture method: the culture bottle rotation speed of 12 to 15 r.p.h.; the infection dosage of 0.01 to 0.001 MLD50/cell; the first 12 hours of the culture at pH = 6.4 to 6.8. Further, pH is maintained at 7.2 to 7.6 for 48 to 72 hours of culture at 37 С, which provides the raw virus production at high infectivity (8.5 to 8.9±0.2 lg MICLD50/cm) and antigenicity (1:128 to 1:256 in PHT and 1:625 to 1:3125 in solid phase ELISA) levels. Phylogenetic analysis of the nucleotide sequences of 3 sites in S and/or M segments showed that it was closely related to strains Smithburn and Entebbe allowing their grouping into a separate cluster. Comparative analysis of nucleotide sequences of genes encoding glycoproteins Gn and Gc, and the nucleoprotein N, shows that most of the substitutions in the sequences of the genes of the RVFV strain 1974-VNIIVViM are synonymous and do not alter the primary amino acid sequence, with insertion and deletion missing. In the nucleotide sequence encoding glycoprotein Gn of strain 1974-VNIIVViM we found 8 substitutions relative to a similar sequence of M segment of a virulent strain Entebbe, and 14 substitutions when compared with M segment of strain Smithburn. A lower (as compared with other attenuated strains) amount of significant amino acid substitutions in RVFV antigenic components was revealed; thus, strain 1974-VNIIVViM selection as a source of immunodominant proteins and/or raw virus for vaccine production seems to be well-grounded.

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

Pax6 Controls the Expression of Critical Genes Involved in Pancreatic α Cell Differentiation and Function*

The paired box homeodomain Pax6 is crucial for endocrine cell development and function and plays an essential role in glucose homeostasis. Indeed, mutations of Pax6 are associated with diabetic phenotype. Importantly, homozygous mutant mice for Pax6 are characterized by markedly decreased β and δ cells and absent α cells. To better understand the critical role that Pax6 exerts in glucagon-producing cells, we developed a model of primary rat α cells. To study the transcriptional network of Pax6 in adult and differentiated α cells, we generated Pax6-deficient primary rat α cells and glucagon-producing cells, using either specific siRNA or cells expressing constitutively a dominant-negative form of Pax6. In primary rat α cells, we confirm that Pax6 controls the transcription of the Proglucagon and processing enzyme PC2 genes and identify three new target genes coding for MafB, cMaf, and NeuroD1/Beta2, which are all critical for Glucagon gene transcription and α cell differentiation. Furthermore, we demonstrate that Pax6 directly binds and activates the promoter region of the three genes through specific binding sites and that constitutive expression of a dominant-negative form of Pax6 in glucagon-producing cells (InR1G9) inhibits the activities of the promoters. Finally our results suggest that the critical role of Pax6 action on α cell differentiation is independent of those of Arx and Foxa2, two transcription factors that are necessary for α cell development. We conclude that Pax6 is critical for α cell function and differentiation through the transcriptional control of key genes involved in glucagon gene transcription, proglucagon processing, and α cell differentiation.

Effects of oil palm phenolics on tumor cells in vitro and in vivo.

The effects of oil palm phenolics (OPP) on cell lines were demonstrated in this study. OPP dosedependent studies in selected cell cultures showed that OPP inhibited the proliferation of mouse IgAsecreting myeloma (J558), estrogen-receptor-positive human breast adenocarcinoma (MCF7) and human lung carcinoma (A549) cells at all doses. However, OPP enhanced the proliferation of Madin Darby Canine Kidney (MDCK) and Aedes albopictus larvae (C6/36) cells at all concentrations, whereas, growth of Syrian Baby Hamster Kidney (BHK) cells was only enhanced with low doses of OPP. 5% OPP in combination with 5% fetal calf serum (FCS) was shown to be as effective as 10% FCS on the growth of BHK and A549 cells. In vivo studies using J558 myeloma cells showed that OPP caused tumor regression. These findings imply that OPP has anti-proliferative effects on tumor cells in vitro and in vivo, but conversely enhances the growth of normal cells.

Pyridine induction of cytochrome P450 1A1, iNOS and metallothionein in Syrian hamsters and protective effects of silymarin

An in vivo assessment for the protective effects of silymarin for pyridine toxicity was investigated through cytochrome P450 isoform CYP1A1 and inducible nitric oxide synthase (iNOS) activity prevention. Moreover, the effect of pyridine-induced oxidative stress on metallothionein I-II (MT), a scavenger of oxygen-derived free radicals, was investigated. Forty Syrian hamsters were allocated into 4 groups. Syrian hamsters were dosed with pyridine (400 mg/kg) intraperitoneally with and without silymarin (200 mg/kg daily by gavage) for 4 days. Pyridine induced diffuse degeneration and necrosis of the proximal and distal renal tubular cells; cloudy swelling, necrosis and hepatocellular atypia of the liver; and degenerative changes in the myocardium. The degree of pathological alterations was less severe with simultaneous silymarin application. CYP1A1, iNOS and MT expression levels were elevated in liver, kidney and heart in response to acute pyridine toxicity. Silymarin application abolished or significantly suppressed the induction of CYP1A1, iNOS and MT expressions in liver, kidney and heart of the pyridine-treated Syrian hamsters. Enhanced synthesis of MT by pyridine possibly implies a purposive cellular response to prevent damage caused by oxygen radicals. However, silymarin significantly reduced the oxidative-stress-inducing effect of pyridine as reflected by decreased synthesis of MT. These results suggest that through oxidant generation, pyridine may cause alteration of the metabolic ways, including nitric oxide-mediated CYP1A1 activity.

Preliminary evaluation of the toxicity of some synthetic furan derivatives in two cell lines and Artemia salina

This study describes the preliminary toxicity evaluation of five new furan derivatives, 2-[2-acetylamino-2-[(benzothiazolyl-substituted)aminocarbonyl]vinyl]-5-nitro furane (compounds A, B, D and E) and 2-[2-phenylamino-2-[benzothiazolylaminocarbonyl]vinyl]furane (compound C). Cytotoxicity was determined using the MTT (tetrazolium salt) method over BHK21 (Syrian baby hamster kidney) and Hep-2 (human larynx carcinoma) cells, which had previously been used to evaluate the cytotoxicity of the 5-nitrofuran derivatives. The lethal concentration 50 (LC(50)) was determined using brine shrimp (Artemia salina) bioassay. Nitrofurantoin was used as reference compound. The results demonstrate that BHK21 cells are more sensitive than Hep-2 cells. This structurally related serial of compounds shows a differential toxicity, which is an indication that the toxicity naturally arising from the nitro group can be modulated by the substituents over the furan ring. Additionally, compound C, the only derivative with no nitro group, was least toxic to Hep-2, but exhibits toxicity to BHK21 cells and brine shrimp. The LC(50 )brine shrimp test (BST) bioassay results were as follows: A, 654.2 microg ml(-1); B, 50.0 microg ml(-1); C, 533.4 microg ml(-1); D, 172.1 microg ml(-1); E, 76.4 microg ml(-1), and NF, >1000 microg ml(-1).

Pax-6 and c-Maf Functionally Interact with the α-Cell-specific DNA Element G1 in Vivo to Promote Glucagon Gene Expression

Specific expression of the glucagon gene in the rat pancreas requires the presence of the G1 element localized at -100/-49 base pairs on the promoter. Although it is known that multiple transcription factors such as Pax-6, Cdx-2/3, c-Maf, Maf-B, and Brain-4 can activate the glucagon gene promoter through G1, their relative importance in vivo is unknown. We first studied the expression of Maf-B, c-Maf, and Cdx-2/3 in the developing and adult mouse pancreas. Although Maf-B was detectable in a progressively increasing number of alpha-cells throughout development and in adulthood, c-Maf and Cdx-2/3 were expressed at low and very low levels, respectively. However, c-Maf but not Cdx-2/3 was detectable in adult islets by Western blot analyses. We then demonstrated the in vivo interactions of Pax-6, Cdx-2/3, Maf-B, and c-Maf but not Brain-4 with the glucagon gene promoter in glucagon-producing cells. Although Pax-6, Cdx-2/3, Maf-B, and c-Maf were all able to bind G1 by themselves, we showed that Pax-6 could interact with Maf-B, c-Maf, and Cdx-2/3 and activate transcription of the glucagon gene promoter. Overexpression of dominant negative forms of Cdx-2/3 and Mafs in alpha-cell lines indicated that Cdx-2/3 and the Maf proteins interact on an overlapping site within G1 and that this binding site is critical in the activation of the glucagon gene promoter. Finally, we show that specific inhibition of Pax-6 and c-Maf but not Cdx-2/3 or Maf-B led to decreases in endogenous glucagon gene expression and that c-Maf binds the glucagon gene promoter in mouse islets. We conclude that Pax-6 and c-Maf interact with G1 to activate basal expression of the glucagon gene.

Aurora A and B Overexpression and Centrosome Amplification in Early Estrogen-Induced Tumor Foci in the Syrian Hamster Kidney: Implications for Chromosomal Instability, Aneuploidy, and Neoplasia

Estrogen-induced Syrian hamster tumors in the kidney represent a useful model to gain insight into the role of estrogens in oncogenic processes. We provided evidence that early tumor foci in the kidney arise from interstitial ectopic uterine-like germinal stem cells, and that early tumor foci and well-established tumors are highly aneuploid (92-94%). The molecular mechanisms whereby estrogens mediate this process are unclear. Here, we report that estrogen treatment induced significant increases in Aurora A protein expression (8.7-fold), activity (2.6-fold), mRNA (6.0-fold), and Aurora B protein expression (4.6-fold) in tumors, compared with age-matched cholesterol-treated kidneys. Immunohistochemistry revealed that this increase in Aurora A and B protein expression was essentially confined to cells within early and large tumor foci at 3.5 and 6 months of estrogen treatment, respectively. Upon estrogen withdrawal or coadministration of tamoxifen for 10 days, a 78% to 79% and 81% to 64% reduction in Aurora A and B expression, respectively, were observed in primary tumors compared with tumors continuously exposed to estrogens. These data indicate that overexpressed Aurora A and B in these tumors are under estrogen control via estrogen receptor alpha. Aurora A coenriched with the centrosome fraction isolated from tumors in the kidney. Centrosome amplification (number and area/cell) was detected in early tumor foci and large tumors but not in adjacent uninvolved or age-matched control kidneys. Taken together, these data indicate that persistent overexpression of Aurora A and B is under estrogen control, and is coincident with centrosome amplification, chromosomal instability, and aneuploidy, and represent an important mechanism driving tumorigenesis.

Towards functional glycomics by localization of binding sites for tissue lectins: lectin histochemical reactivity for galectins during diethylstilbestrol-induced kidney tumorigenesis in male Syrian hamster

Endogenous lectins act as effectors of cellular activities such as growth regulation, migration, and adhesion. Following their immunohistochemical localization in our previous study (Saussez et al. in Histochem Cell Biol 123:29-41, 2005) we purified several galectins and used them as tools for monitoring accessible binding sites. Herein, we report the use of galectin histochemistry for the analysis of diethylstilbestrol (DES)-induced renal tumors in male Syrian hamster kidney (SHKT). Sections of normal kidney and DES-treated kidney were analyzed with biotinylated galectins-1, -3 (full-length and truncated), and -7. Accessible binding sites were detected, localization was predominantly extracellular and confined to medium-sized and large tumors. Monitoring the SHKT-derived HKT-1097 line, processed in vitro or as xenograft material, cytoplasmic and nuclear staining for galectins-1, -3, and -3tr could be observed. Adaptation of SHKT cells to long-term growth in culture is thus associated with emergence of this signal. Our data set illustrates the feasibility to complement immunohistochemical data by application of the tissue lectins as probes, and to detect regulation of galectin reactivity with differential characteristics within tumor progression in vivo and unique features of the tumor cell line in vitro and in vivo.