scholarly journals Processing of Human Nerve Growth Factor β Proprotein in the Plant Cell Apoplast

Author(s):  
Maryam Zangi ◽  
Hamideh Ofoghi ◽  
Parastoo Ehsani ◽  
Fazel Shokri ◽  
Somayeh Ghotloo ◽  
...  

Abstract The Human HNGF-β gene encodes a pre-pro-NGF-β precursor protein. The signal peptide is cleaved off in the endoplasmic reticulum and the resulting pro-protein is processed in the trans-Golgi network by the Furin enzyme.The processing of the proproteins in the plant cells is not clearly defined. In the present study, various changes were applied to the HNGF-β gene sequence to increase its expression in the plant cells. The construct containing the synthesized Plant NGF-β gene was agro-infiltrated into the Nicotiana benthamiana leaves. Thereafter, the expression of various forms of the recombinant NGF-β and its processing was evaluated using dot blot, western blot, and RP-HPLC analyses. Finally, the biological activity of the mature NGF-β purified from the apoplast was assessed on the differentiation of PC12 cells and the expression of tetanus toxin receptors on their surfaces.Dot blot and western blot results showed that the total soluble proteins extracted from the plant leaves and the apoplast extract contained pro-NGF-β and mature NGF-β proteins, respectively. The RP-HPLC results reconfirmed the presence of the mature NGF-β in the apoplast extract. The amount of the mature NGF-β produced in the plant leaves was estimated to be about 39µg/g of the fresh leaves. A gradual increase in the length and number of the neurites of the differentiated PC12 cells was showed upon treatment with the mature NGF-β. Immunofluorescence experiments showed that the FITC-labeled tetanus toxin strongly bound to PC12 cells treated with mature NGF-β. This modification shows a very important advantage for plants to produce valuable biosimilar pharmaceutical proteins from precursor without applying biochemical or co-expression of modifying enzymes.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.


2006 ◽  
Vol 173 (2) ◽  
pp. 241-251 ◽  
Author(s):  
Malika Ahras ◽  
Grant P. Otto ◽  
Sharon A. Tooze

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.


2021 ◽  
Author(s):  
Renata Kasprzyk ◽  
Tomasz J. Spiewla ◽  
miroslaw smietanski ◽  
Sebastian Golojuch ◽  
Laura Vangeel ◽  
...  

SARS-CoV-2, the cause of the currently ongoing COVID-19 pandemic, encodes its own mRNA capping machinery. Insights into this capping system may provide new ideas for therapeutic interventions and drug discovery. In this work, we employ a previously developed Py-FLINT screening approach to study the inhibitory effects of compounds against the cap guanine N7-methyltransferase enzyme, which is involved in SARS-CoV-2 mRNA capping. We screened five commercially available libraries (7039 compounds in total) to identify 83 inhibitors with IC50 < 50 μM, which were further validated using RP HPLC and dot blot assays. Novel fluorescence anisotropy binding assays were developed to examine the targeted binding site. The inhibitor structures were analyzed for structure-activity relationships in order to define common structural patterns. Finally, the most potent inhibitors were tested for antiviral activity on SARS-CoV-2 in a cell based assay<br>


2022 ◽  
Author(s):  
Jing Wu ◽  
zhonghao li ◽  
xiaoke dong ◽  
siyuan yuan ◽  
jinmin liu ◽  
...  

Abstract Background: Acute ischemic stroke (AIS) and following reperfusion therapy-induced cerebral ischemia reperfusion (I/R) injury have been recognized as an important subject of cerebrovascular disease with high mortality. Oxidative stress is an important pathological process of cerebral I/R injury. microRNA-19a (miR-19a) is involved in I/R. As the organ protectant agent, Shenmai Injection (SMI) is widely used in the clinical treatment of cerebral infarction. Purpose: This study aims to explore whether SMI can reduce oxidative stress by regulating miR-19a, thereby treating I/R injury. Methods: The oxidative stress state of PC12 cells was induced by H2O2, and then the cells were cultured with SMI. The therapeutic effect of SMI was evaluated by detecting cellular superoxide dismutase (SOD), malondialdehyde (MDA) and other oxidative markers with the kit. Western blot, PCR, immunofluorescence and other techniques were used to elucidate the potential mechanism of SMI. Results: Cell viability assay results showed that SMI could improve the viability of PC12 cells stimulated by H2O2. Compared with the H2O2 group, after SMI treatment, the contents of MDA and reactive oxygen species (ROS) were significantly reduced, while the activity of SOD was significantly increased, and SMI could reduce apoptosis by increasing the content of adenosine 5'-triphosphate (ATP) in cells and enhancing the mitochondrial membrane potential (∆Ψm). Western blot and qRT-PCR results showed that these effects were partially achieved through the AMPK/Sirt1/PGC-1α pathway. The level of miR-19a was significantly increased in H2O2 group, and SMI could protect the cells by reducing miR-19a. Further investigated the target of miR-19a, and transfected cells with miR-19a mimic and inhibitor respectively. We found that AdipoR2 was a direct target of miR-19a, and miR-19a could inhibit AdipoR2/PI3K/Akt/mTOR pathway. Conclusion:SMI can activate AMPK/Sirt1/PGC-1α and AdipoR2/PI3K/Akt/mTOR pathways by reducing miR-19a levels, and protect PC12 cells stimulated by H2O2.


2002 ◽  
Vol 177 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Shahram Barati ◽  
Fariba Chegini ◽  
Plinio Hurtado ◽  
Robert A. Rush

2009 ◽  
Vol 60 (5) ◽  
pp. 865-881 ◽  
Author(s):  
Sheung Kwan Lam ◽  
Yi Cai ◽  
Yu Chung Tse ◽  
Juan Wang ◽  
Angus Ho Yin Law ◽  
...  

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4623-4623
Author(s):  
Kathy L McGraw ◽  
Ashley A Basiorka ◽  
Joseph Johnson ◽  
Justine Clark ◽  
Gisela Caceres ◽  
...  

Abstract Erythropoietin receptor (EpoR) signaling is impaired in patients with Myelodysplastic Syndromes (MDS) despite appropriate growth factor production and cellular receptor display. We previously reported that EpoR signaling is dependent upon receptor localization within membrane lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity (McGraw KL, et al. PLoS One 2012). Here, we show that MDS erythroid progenitors display markedly diminished raft assembly (p=0.005) and smaller raft aggregates (p=0.023) compared to normal controls. Because lenalidomide triggers raft coalescence in T-lymphocytes to promote immune synapse formation, we assessed the effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lipid rafts were isolated from UT7 cells using ultracentrifugation and identified by GM-1 dot blot and Lyn kinase western blot. Lenalidomide rapidly induced lipid raft formation in UT7 cells which was confirmed by confocal microscopy visualization of GM-1 fluorescence. Lenalidomide also significantly induced lipid raft formation in pooled MDS erythroid progenitors (CD71+, cKit+) from 11 patients [mean raft size, control (n=569) vs. lenalidomide treatment (n=659), p<0.001], with no significant change observed in pooled erythroids from 3 normal donors (n= 327 for control and n=365 for lenalidomide treated, p=0.37). Interestingly, lipid rafts were significantly larger in erythroid progenitors from patients who responded (n=5) to lenalidomide treatment compared to non-responders (n=3) (75.52 ±13.68 vs. 35.85 ±8.56, p=0.02). Although lenalidomide increased raft size in erythroid progenitors from both responders (p=0.0007) and non-responders (p=0.013), mean raft size was greater in erythroid precursors from responding patients after treatment (p=0.11). Increased raft aggregation after lenalidomide treatment was accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase, whereas the JAK2 phosphatase, CD45, a negative regulator of EpoR signaling, was displaced from raft fractions. Incubation with lenalidomide prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 cells and primary MDS erythroid precursors. Bone marrow specimens from 12 non-del(5q) IPSS lower risk, lenalidomide naive MDS patients were analyzed by flow cytometry to compare changes in STAT5 phosphorylation in response to Epo stimulation in the presence or absence of lenalidomide. We found a 79.1% mean increase in p-STAT5 mean fluorescence intensity (MFI 95th percentile) in CD45dim, CD71high, Glylow erythroid precursors in 7 of the 12 patient specimens following lenalidomide exposure. Furthermore, increased STAT5 phosphorylation was accompanied by increased DNA binding of the transcription factor in UT7 cells, and improved erythroid colony forming capacity in both UT7 and primary MDS bone marrow cells. Raft induction was associated with F-actin polymerization that was blocked by Rho kinase inhibition and confirmed by lipid raft isolation followed by dot blot with western blot and confocal microscopy. These data provide new insight into abnormalities in the EpoR signaling platform that underlie impaired Epo responsiveness in MDS erythroid precursors. Our findings that deficient raft integrity impairs EpoR signaling provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS. These data also warrant investigation in a larger data set to determine whether lipid raft size may be a predictive biomarker for lenalidomide response. Disclosures List: Celgene: Consultancy.


2001 ◽  
Vol 13 (2) ◽  
pp. 287 ◽  
Author(s):  
Dae Heon Kim ◽  
Young-Jae Eu ◽  
Cheol Min Yoo ◽  
Yong-Woo Kim ◽  
Kyeong Tae Pih ◽  
...  

2001 ◽  
Vol 8 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Paul T. Fawcett ◽  
Carlos D. Rose ◽  
Sandra M. Budd ◽  
Kathleen M. Gibney

ABSTRACT This study evaluated the effects of vaccination with OspA on the use of serologic tests as aids in the diagnosis of Lyme borreliosis. Sera from control and OspA-immunized mice and from OspA-immunized human volunteers were tested for serologic reactivity to Borrelia burgdorferi. Testing was performed with samples obtained prior to administration of vaccine and at 30 days following administration of an initial and a second dose of OspA vaccine. The assays used to assess serologic reactivity included an in-house-developed enzyme-linked immunosorbent assay (ELISA), an in-house-developed Western blot assay, two commercial Western blot tests, and a commercially available dot blot assay. Data obtained from this study demonstrate that immunization with the OspA vaccine will cause ELISA to yield positive results (as reported previously) for the majority of vaccine recipients. Results obtained from Western blot analysis indicate that vaccination with recombinant OspA induces production of antibodies which bind to several different borrelial proteins. The degree of reactivity detected by Western blotting varied greatly between the three assays used. The in-house assay showed the least reactivity, while one commercial Western blot test actually yielded positive test results for infection with B. burgdorferi. The usefulness of all three Western blot assays for the diagnosis of potential infection in a vaccine recipient is severely limited by the extensive reactivity caused by vaccination alone. Antibodies produced in response to OspA vaccination did not significantly affect the performance of the dot blot test; thus, it could provide a reliable means to test for infection withB. burgdorferi in OspA vaccine recipients.


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