Comparative sensitivity analysis between two methods for species differentiation and interspecies cross contamination in animal cell culture

Background: The present article focuses on the design and development of a highly sensitive and convenient approach for rapid detection of animal species and cross contaminations quickly during cell cultures as most important document for manufacturing working cell bank system. This test is one of the four most important documents during implementing the banking system. By using this modified test, one of the major risks in cell culture laboratories, cross- contamination and misidentifications with microorganisms of cell lines will also be important to be confirmed

Materials and Methods: A PCR _RFLP assay was optimized based on the use of a pair of primers that anneal to a portion the cytochrome b gene in all the species. The amplification product was digested with a panel of six restriction enzymes and the pattern derived was resolved on 3% high resolution agarose gel for 2 species, human and primate. As a control test iso enzyme assay as a conventional method was used

Results: This protocol produced a unique restriction pattern and the origin was confirmed by this analysis. The sensitivity in detecting interspecies cross contamination was at least 100 pg DNA/reaction, which was sufficient for detection of each species of DNA

Conclusion: The method developed in this study will provide a useful tool for the authentication of animal species and is also more comparable and time consuming, compared with conventional analysis. Using this method, significant differences between human and non-human as well as cross- contamination between different cell lines are simply distinguished

In vivo and in vitro anti-angiogenesis effect of venom-derived peptides [ICD-85]

Agiogenesis is the development of new blood vessels from pre-existing vasculatures. Although essential in the physiological process, it becomes pathological in various diseases including cancer. Preventing the formation of new blood vessels causes reductions in tumor size and metastasis. This study has been undertaken to elucidate the anti-angiogenesis effects of ICD-85 [derived peptides from venom]. We evaluated the ICD-85 anti-angiogenesis activity by the in vivo CAM assay and in vitro tube formation assay of human umbilical vein endothelial cells [HUVECs]. The anti-proliferative activity of ICD-85 was also determined through MTT assay on HUVECs. Results of this study revealed the anti-proliferative activity of ICD-85 on the HUVEC cell line with an IC50 of 12 micro g/mL. The in vivo CAM assay also clearly showed the prevention of new vascular formation when the chick embryos were exposed to 0.15 micro g/disc of ICD-85. In vitro tube formation assay of HUVECs also showed the complete prevention of capillary tube formation on 18 micro g/mL. Based on the results obtained in this study, ICD-85 has anti-angiogenesis activity as shown by the prevention of capillary tube formation and the CAM assay

Cytotoxic effect of ICD-85 [venom-derived peptides] on HeLa cancer cell line and normal LK cells using MIT assay

Cancer is the fifth leading cause of death worldwide. There are considerable efforts to identify naturally occurring substances for use as new drugs in cancer therapy. Some components of animal venoms have been identified that possess substantial anticancer properties. In our previous studies, the cytotoxic effects of ICD-85 [venom-derived peptides] have been reported on HL-60 and MDA-MB231 cell lines. This has prompted us to investigate the comparative cytotoxic effects of ICD-85 on the HeLa cell line and normal lamb kidney [LK] cells. Cells were exposed to various concentrations [8 x 10[4] to 5.6 x 10 microg/mI] of ICD-85 at various incubation times [24, 48 and 72 hours]. Cell viability was measured by the MTT assay. A morphological study was also carried out using an inverted microscope. Caspase-8 activity was assayed by the Caspase-8 Colorimetric Assay Kit in HeLa cells that were exposed to ICD-85 for 48 hours. Data analysis showed that ICD-85 has a dose-dependent cytotoxic effect on HeLa cells with an inhibitory concentration 50% [IC[50]] of 26.62 +/- 2.13 microg/mI at 24 hours, 27.33 +/- 2.35 microg/mI at 48 hours, and 28.13 +/- 2.52 microg/mI at 72 hours. Results also indicated that the cytotoxic effect of ICD-85, at 48 and 72 hours incubation times did not show significant alteration compared to 24 hours of exposure. Interestingly, the minimum concentration of ICD-85 which showed a cytotoxic effect on LK cells was found to be 3500-fold less than the minimum concentration that showed a cytotoxic effect on the HeLa cancer cells. While morphological analysis revealed a significant difference that included the characteristic rounding of dying cells by treatment with ICD-85 compared with untreated HeLa cells, this difference was not observed in normal cells. ICD-85 increased caspase-8 activity in HeLa cells after 48 hours of exposure. ICD-85 has a dose-dependent cytotoxic effect on HeLa cancer cells in contrast with its negligible effect on normal LK cells

Induction of apoptosis in human leukemia cell line [HL60] by animal's venom derived peptides [ICD-85]

Our previous studies revealed an inhibitory effect of ICD-85 [Venom derived peptides] on breast cancer cell line MDA-MB231. ICD-85 was also confirmed by in-vivo studies to suppress the breast tumor in mice. However, the exact mechanism of ICD-85 was unknown. Hence, the present study was undertaken to assess the mechanism of ICD-85 effect as an anti-proliferative agent of cancer cells. The effect of ICD-85 on proliferation of HL-60 cancer cells was determined by using the MTT assay. The morphological changes of ICD-85 treated HL-60 cells were observed under transmission electron microscope [TEM] DNA fragmentation analysis was also carried out using gel electrophoresis. ICD-85 induced the marked inhibition of HL60 cell proliferation with an IC[50]-value of 0.04 |ug/mL following 24 h of incubation. ICD-85 treated cells when compared with untreated cells, showed nuclear material condensation, endoplasmic reticulum dilation, mitochondria swelling or degradation, increased cytoplasmic vacuoles, reduction or disappearance in cytoplasmic process and decreased nuclear/cytoplasmic ratio was observed. The characteristic DNA ladder formation of ICD-85-treated cells in agarose gel electrophoresis confirmed the results obtained through the electron microscopy. The results of the present study indicated that ICD-85 inhibited the cancer cell proliferation by inducing cell apoptosis

Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3Dpol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3Dpol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp26-->Glu substitution in a beta sheet located within a small groove of the 3Dpol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.

Serological study of bovine viral diarrhea virus [BVDV] infection in water buffalo [Bubalusbubalis] in Ahvaz in the southwestern region of Iran

A serological survey was carried out to determine the prevalence rate of bovine viral diarrhea virus [BVDV] infections in water buffalo [Bubalusbubalis] in Ahvaz, which is the center of the Khouzestan province in Iran. For this purpose, blood samples were taken from 310 slaughtered buffaloes at the abattoir. Sera were tested via the serum neutralization test. Serum neutralization was performed by National Animal Diseases Laboratory [NADL], in order to isolate the genotype 1 strain of bovine viral diarrhea virus. The results indicate that 105 [33.9%] buffaloes had antibodies to BVDV. The prevalence of infection in females and males were 39.5% and 22.78%, respectively, and statistical analysis showed that this difference was significant. Although there was a non-significant difference between heifers and males, the difference between cows and bulls was highly significant

Sequence and phylogenetic analysis of the non-structural 3A and 3B protein-coding regions of foot-and-mouth disease virus subtype A Iran 05

The A Iran 05 foot-and-mouth disease virus (FMDV) subtype was detected in Iran during 2005 and has proven to be highly virulent. This study was undertaken to focus on molecular and phylogenetic analysis of 3A and 3B coding-regions in the A Iran 05 field isolate. To assess the genetic relatedness of A Iran 05 isolate the nucleotide and predicted amino acid sequences of the 3AB region of type A FMDV isolates were compared with twenty previously described type A FMDV isolates. The phylogenetic tree based on the 672 bp 3AB gene sequences of type A FMDV from thirteen different locations clustered them into five distinct lineages. The A Iran 05 isolate clustered in lineage A along with four type A variants and was closely matched with viruses isolated in Turkey and Pakistan during 2005~2006. The number of protein sequence differences exhibited by each of the isolates revealed that A Iran 05 isolate contains three amino acid substitutions at positions 47 and 119 of 3A and 27 of the 3B coding region. The nucleotide identity between A Iran 05 and the other four isolates of lineage A was estimated to be 98%.