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Plant- Made Trastuzumab (Herceptin) Inhibits HER2/Neu+ Cell Proliferation and Retards Tumor Growth. Abstract. Background. Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (m. Ab), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the m.
HC Verma Solutions Download Solution of HC Verma Concept of Physics Part 1 and Part 2 are given here. HC Verma Solutions are intended for students who are solving the. Hillary: rolling stones: cecilia bartoli: marcos aguinis: rachel mcadams: ridley scott: grisham: kobe bryant: leopoldo lopez: leonardo di caprio: miquel barcelÓ. Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer.
Ab. Here we used an Agrobacterium- mediated transient expression system to produce trastuzumab in plant cells. Methodology/Principal Findings. We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron- optimized Tobacco mosaic virus- and Potato virus X- based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full- size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK- BR- 3, in fluorescence- activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER- 2- expressing cancer cell proliferation. We show that plant- made trastuzumab (PMT) bound to the Her. SK- BR- 3 cells and efficiently inhibited SK- BR- 3 cell proliferation.
Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her. Conclusions/Significance. We conclude that PMT is active in suppression of cell proliferation and tumor growth. Introduction. There was a time when most medicinal compounds were simply extracted from plants, but now, plant molecular biology produces valuable recombinant pharmaceutical molecules, including enzymes, vaccines, and antibodies [1]–[9]. Such “molecular farming” has many economic and qualitative benefits, including reduced health risks from human and animal pathogen contamination and comparatively high yields.
It has been estimated that the cost of pharmaceutical protein production in plants could be 1. Plants rapidly accumulate single- chain [1. Plants may be a source of biosimilars, new versions of known pharmaceuticals, including anticancer antibodies [2. Human epidermal growth factor receptor 2 (HER2/neu) is an oncogene involved in abnormal cell growth in breast cancer and is a target for the humanised monoclonal antibody (m.
Ab) trastuzumab (Herceptin) [2. US Food and Drug Administration for the treatment of HER2/neu- overexpressing breast tumours. HER2/neu is overexpressed in 2. Trastuzumab induces antibody- dependent cellular cytotoxicity (ADCC), inhibits HER2- mediated signaling, and prevents cleavage of the extracellular domain of HER2 [2. In HER2- positive breast cancer, trastuzumab has shown a survival advantage in early and metastatic disease and is now the standard of care [2.
Trastuzumab is produced by recombinant DNA technology in a mammalian cell (Chinese Hamster Ovary) culture. Recently, the production of plant- made trastuzumab [PMT] was shown in plant using the magn.
ICON viral- based transient expression system [1. Functional assays revealed that plant- produced trastuzumab and Herceptin have similar antiproliferative effects in vitro on HER2+ breast cancer cells. Here, we used also genes encoding both heavy and light chains of trastuzumab, cloned into 3. S- and virus- based vectors and expressed in Nicotiana benthamiana leaves. We show that both vector systems result in high yield of full- size antibodies, PMT, which recognizes HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK- BR- 3, and active in suppression of cell proliferation in vitro.
Moreover, mouse PMT administration retarded efficiently the growth of xenografted Her. Results. Accumulation and purification of assembled PMT in N. To prove the applicability of our plant transient system for the production of anticancer m. Ab, we synthesized genes encoding the heavy and light chains of the trastuzumab protein using the amino acid sequence published in Drug. Bank (accession number DB0. S- based vectors (3.
S- LC and 3. 5S- HC) (Figure 1. A). N. benthamiana leaves co- agroinjected with PT- LC, PT- HC and the silencing suppressor Tomato Bushy Stunt Virus (TBSV) p. PMT, as revealed in a gel stained with Coomassie blue. Assembled antibodies were extracted from plant tissue, purified on protein A affinity columns, and analyzed either by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE) under reducing conditions followed by Coomassie blue staining (Figure 1.
B) or by western blotting probed with gamma- HC- and kappa- LC- specific antibodies (Figure 1. C,D). Bands corresponding to the heavy chain (∼5. Da) and the light chain (∼2.
Da) are clearly visible on the Coomassie- stained gel (Figure 1. B) and on western blots (Figure 1.
C, D). Expression of 3. S- based constructs was maximal at 3 dpi, and the yield was between 1. FW), depending on the experiment. Production of assembled PMT in N. S- based light- and heavy- chain- expressing vectors. Next, PMT light and heavy chain genes were cloned into PVX- based and TMV- based vectors, respectively (Figure 2. A), as these vectors are able to replicate within the same cell with high efficiency and do not compete with each other for replication binding sites [1.
Fully assembled PMT was extracted from N. HC- TMV and LC- PVX vectors at 7 dpi when the maximal level of antibody production was detected (data not shown).
Antibodies were purified on protein A sepharose columns and analyzed via SDS- PAGE under non- reducing (Figure S1. A) or reducing (Figure S1. B) conditions. MALDI- TOF analysis showed an identical peptide composition of PMT and trastuzumab light and heavy chains (data not shown).
Assembled PMT is detected on gels stained with Coomassie blue. Western blot analysis was performed to determine the composition of the other bands on the gel.
Probing with anti- gamma- chain antibodies revealed two high molecular weight bands, also detected with anti- kappa- chain antibodies (Figure 2. B,C), that likely represent fully assembled Ig. G molecules and heterotrimers [(HC)2+LC].
The band that corresponds to the monomeric heavy chain is also visible in Figure 2. B. Of these forms, the heterotetramer [(HC)2+(LC)2] is the most intense band visible after Coomassie blue staining (Figure S1.
B). Another band (∼9. Da) detected on both 2. B and 2. C western blots appears to be a heterodimer of heavy and light chains. In addition, a strong band most likely corresponding to the dimeric form of the light chain (∼4. Da) was produced with anti- kappa- chain antibodies.
After treatment with 2- mercaptoethanol, all additional bands disappeared, with only heavy (Figure 2. D) and light chains (Figure 2. E) present. The yield of PMT expressed from viral vectors was between 2.
FW depending on the experiment. Accumulation and purification of assembled PMT in N. PVX- based and heavy- chain- encoding TMV- based vectors.
Further PMT purification on an AKTApurifier (GE Healthcare) was used to obtain assembled PMT that was free of additional complexes between heavy and light chains (Figure S2). Figure 2. F shows capillary electrophoresis of PMT performed on an Agilent 2. Bioanalyzer under reducing conditions, where peak 1. HC and peak 8 corresponds to LC. It is likely that peaks 2 and 3 are low molecular products of PMT degradation.
Direct comparison of PMT and trastuzumab revealed a similar protein profile on gels stained with Coomassie blue (Figure 2. G, H) and the absence of visible contaminations on HPLC trace analysis (Figure 2.
I). PMT recognises a HER2/neu peptide mimotope. Trastuzumab binds amino acids 5. C- terminal end of domain IV of the extracellular region of HER2 [2. Recently, the conformational epitope 5. HER- 2/neu [3. 0]. To examine whether PMT may bind the trastuzumab conformational epitope 5. CYC [2. 9], [3. 0] and compared PMT and trastuzumab binding by ELISA.
Polystyrene plates were coated overnight with the 5. CYC peptide and probed with PMT and trastuzumab the following day.
Figure 3 shows that both m. Abs, PMT and trastuzumab, bind the synthetic peptide 5. CYC in a dose- dependent manner. Binding of PMT to a HER2/neu peptide mimotope.
PMT binds efficiently to HER2/neu- expressing SK- BR- 3 cells. For quantitative estimation of the binding affinity of PMT to Her. FACS analysis was performed. Figure 4 (D–F) shows a high percentage (7. PMT binding to surface HER2/neu independently of antibody concentration. This result is similar to the data obtained using trastuzumab (Figure 4.
A–C). Examination of PMT binding to HER2/neu. Next, immunocytochemical staining of a human mammary gland adenocarcinoma cell line that overexpresses HER2/neu, SK- BR- 3, was performed to test the functional activity of the plant- made m. Ab. PMT bound to Her. A0. 48. 5 (Dako, Denmark) (data not shown).
The same result was obtained on tissue samples from a patient with Her. We conclude that PMT and trastuzumab exhibit no difference in binding capacity for HER2/neu. PMT inhibits SK- BR- 3 cell growth in vitro. The SK- BR- 3 cell line was used to compare the antiproliferative properties of PMT and trastuzumab. Varying concentrations of PMT (0.
MTT assays. The data presented in Figure 5 show similar inhibitory effects of PMT and trastuzumab on SK- BR- 3 cell proliferation. We conclude that PMT possesses the anticancer properties of trastuzumab.
Effects of PMT on growth of the breast cancer cell line SK- BR- 3 in MTT assays. PMT retards SKOV3- derived tumor growth in a xenograft mouse model of human ovarian cancer. Having shown that PMT suppresses tumor cell growth, we investigated its antitumor effects in SKOV3 Her. Although it is known that SKOV3- derived tumors are less sensitive to trastuzumab than are SK- BR- 3- derived tumors [3. PMT. As shown in Figure 6, PMT treatment caused a delay in tumor growth. After 8 consecutive injections (1. Material and Methods), the reduction in tumor growth was 7.
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