The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10

2006 ◽  
Vol 63 (11) ◽  
pp. 681-695 ◽  
Author(s):  
Percy Bondallaz ◽  
Anne Barbier ◽  
Sophia Soehrman ◽  
Gabriele Grenningloh ◽  
Beat M. Riederer
Keyword(s):  
Transfected Cells ◽  
Microtubule Stability

scholarly journals Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA.

1997 ◽  
Vol 17 (4) ◽  
pp. 1938-1946 ◽  
Author(s):  
D Aviezer ◽  
R V Iozzo ◽  
D M Noonan ◽  
A Yayon
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Core Protein ◽  
Critical Role ◽  
Receptor Activation ◽  
Fibroblast Growth ◽  
Proliferating Cells ◽  
Transfected Cells ◽  
High Affinity

Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.

State of the art in the production of transgenic goats

2004 ◽  
Vol 16 (4) ◽  
pp. 465 ◽  
Author(s):  
H. Baldassarre ◽  
B. Wang ◽  
C. L. Keefer ◽  
A. Lazaris ◽  
C. N. Karatzas
Keyword(s):  
Recombinant Proteins ◽  
Traditional Method ◽  
Nuclear Transfer ◽  
State Of The Art ◽  
Expression Vectors ◽  
Marketing Approval ◽  
Transfected Cells ◽  
Pronuclear Microinjection ◽  
Transgenic Goats

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

scholarly journals MACC1 Contributes to the Development of Osteosarcoma Through Regulation of the HGF/c-Met Pathway and Microtubule Stability

2020 ◽  
Vol 8 ◽  
Author(s):  
Jia Wen ◽  
Yi Xie ◽  
Yingqiang Zhang ◽  
Jiazhen Li ◽  
Jiaping Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumor Growth ◽  
Signaling Pathway ◽  
Normal Tissues ◽  
Microtubule Stability ◽  
Promising Therapeutic Target ◽  
Tumor Growth And Metastasis ◽  
Hepatocyte Growth ◽  
Proliferative Ability

Osteosarcoma (OS) is the most prevalent human bone malignancy, and presents a global annual morbidity of approximately five cases per million. Notably, precise and efficient targeted therapy has become the most promising strategy for the treatment of OS; however, there is still an urgent need for the identification of suitable therapeutic targets. Metastasis-associated in colon cancer 1 (MACC1) was first identified in colon tumors by differential display RT-PCR, and was shown to be involved in the regulation of colon tumor growth and metastasis through the hepatocyte growth factor (HGF)/c-Met signaling pathway. Additionally, MACC1 overexpression has been reported to induce the growth of several types of cancers, including glioblastoma multiforme and gastric cancer. However, whether MACC1 also plays a role in the progression of OS remains unclear. In this study, we found that MACC1 was highly expressed in human OS tissues, as well as in U-2OS and MG-63 cells, when compared with normal tissues and osteoblasts, respectively. Our data further indicated that MACC1 expression was correlated with several clinicopathological features of OS. Through in vitro assays, we found that MACC1 depletion markedly suppressed the proliferative ability of both OS cells and endothelial cells, and inhibited the angiogenic capacity of endothelial cells. Similarly, MACC1 depletion inhibited tumor growth, metastasis, and angiogenesis in mice. Mechanistically, we found that MACC1 could bind to the MET promoter, and enhanced the proliferation of both OS cells and endothelial cells through the HGF/c-Met signaling pathway. Furthermore, we show that MACC1 also promoted angiogenesis by regulating microtubule dynamics, thereby promoting the progression of OS. Our results indicate that MACC1 may be a new and promising therapeutic target for the treatment of OS.

scholarly journals Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

2019 ◽  
Vol 12 (594) ◽  
pp. eaaw2939 ◽  
Author(s):  
Rozita Adib ◽  
Jessica M. Montgomery ◽  
Joseph Atherton ◽  
Laura O’Regan ◽  
Mark W. Richards ◽  
...  
Keyword(s):  
Double Mutant ◽  
Constitutive Activation ◽  
Mitotic Kinases ◽  
Terminal Domain ◽  
Microtubule Stability ◽  
Cryo Electron Microscopy ◽  
Chromosome Congression ◽  
Spindle Microtubules ◽  
Mitotic Phosphorylation

EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo–electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.

scholarly journals Human C5-specific single-chain variable fragment ameliorates brain injury in a model of NMOSD

2019 ◽  
Vol 6 (3) ◽  
pp. e561 ◽  
Author(s):  
Wenli Zhu ◽  
Zhen Wang ◽  
Suying Hu ◽  
Ye Gong ◽  
Yuanchu Liu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Phage Display ◽  
Membrane Attack Complex ◽  
Single Chain ◽  
Inflammatory Infiltration ◽  
Transfected Cells ◽  
Complement Dependent Cytotoxicity ◽  
Test Specificity

ObjectiveUsing phage display, we sought to screen single-chain variable fragments (scFvs) against complement C5 to treat neuromyelitis optica spectrum disorder (NMOSD).MethodsAfter 5 rounds of phage display, we isolated individual clones and identified phage clones specifically binding to C5 using ELISA. Using aquaporin-4 (AQP4)-transfected cells in vitro, we confirmed whether these scFvs prevented complement-dependent cytotoxicity (CDC) caused by the serum of patients with NMOSD and human complement (hC). We selected an NMOSD mouse model, in which intracerebral NMOSD immunoglobulin G (IgG) and hC injections induce NMOSD-like lesions in vivo.ResultsWe obtained scFvs to test specificity and blocking efficiency. The scFv C5B3 neutralized C5 in the complement activation pathway, which prevented AQP4-IgG–mediated CDC in AQP4-transfected cells. In an NMOSD mouse model, C5B3 prevented AQP4 and astrocyte loss, decreased demyelination, and reduced inflammatory infiltration and membrane attack complex formation in lesions.ConclusionsWe used phage display to screen C5B3 against C5, which was effective in inhibiting cytotoxicity in vitro and preventing CNS pathology in vivo.

scholarly journals Transient Silencing of a Type IV P-Type ATPase,Atp10c, Results in Decreased Glucose Uptake in C2C12 Myotubes

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
S. E. Hurst ◽  
S. C. Minkin ◽  
J. Biggerstaff ◽  
M. S. Dhar
Keyword(s):  
Type 2 Diabetes ◽  
Glucose Uptake ◽  
Glucose Transporter ◽  
Mapk Pathway ◽  
Specific Inhibitor ◽  
C2c12 Myotubes ◽  
Glucose Transporter 1 ◽  
Transfected Cells

Atp10cis a strong candidate gene for diet-induced obesity and type 2 diabetes. To identify molecular and cellular targets of ATP10C,Atp10cexpression was alteredin vitroin C2C12 skeletal muscle myotubes by transient transfection with anAtp10c-specific siRNA. Glucose uptake assays revealed that insulin stimulation caused a significant 2.54-fold decrease in 2-deoxyglucose uptake in transfected cells coupled with a significant upregulation of native mitogen-activated protein kinases (MAPKs), p38, and p44/42. Additionally, glucose transporter-1 (GLUT1) was significantly upregulated; no changes in glucose transporter-4 (GLUT4) expression were observed. The involvement of MAPKs was confirmed using the specific inhibitor SB203580, which downregulated the expression of native and phosphorylated MAPK proteins in transfected cells without any changes in insulin-stimulated glucose uptake. Results indicate thatAtp10cregulates glucose metabolism, at least in part via the MAPK pathway, and, thus, plays a significant role in the development of insulin resistance and type 2 diabetes.

scholarly journals Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4

2005 ◽  
Vol 16 (10) ◽  
pp. 4648-4659 ◽  
Author(s):  
Brandon E. Kremer ◽  
Timothy Haystead ◽  
Ian G. Macara
Keyword(s):  
Mammalian Cells ◽  
Spectroscopic Analysis ◽  
Molecular Function ◽  
Rich Region ◽  
Intact Cells ◽  
Pronounced Increase ◽  
Binding Partner ◽  
Microtubule Stability ◽  
Proline Rich Region

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

scholarly journals TGF-beta 1 is an autocrine-negative growth regulator of human colon carcinoma FET cells in vivo as revealed by transfection of an antisense expression vector.

1992 ◽  
Vol 116 (1) ◽  
pp. 187-196 ◽  
Author(s):  
S P Wu ◽  
D Theodorescu ◽  
R S Kerbel ◽  
J K Willson ◽  
K M Mulder ◽  
...  
Keyword(s):  
Cell Growth ◽  
Colon Carcinoma ◽  
Growth Regulator ◽  
Human Colon ◽  
Tgf Beta ◽  
Transfected Cells ◽  
Colon Carcinoma Cells ◽  
Human Colon Carcinoma

Transforming growth factor-beta 1 (TGF-beta 1) has previously been implicated as a potential negative autocrine or paracrine growth regulator of certain cell types (Arteaga, C. L., R. J. Coffey, Jr., T. C. Dugger, C. M. McCutchen, H. L. Moses, and R. M. Lyons. 1990. Cell Growth & Differ. 1:367-374; Hafez, M. M., D. Infante, S. Winawer, and E. Friedman. 1990. Cell Growth & Differ. 1:617-626; Glick, A. B., K. C. Flanders, D. Danielpour, S. H. Yuspa, and M. B. Sporn. 1989. Cell Regulation. 1:87-97). This is based mainly on experiments assessing the effects of exogenous TGF-beta 1 or neutralizing antibodies to TGF-beta 1 on normal or tumor cell proliferation in vitro. However, direct evidence demonstrating such a negative regulation of tumor cell growth in vivo is still lacking. To overcome this problem we have constructed and used an antisense expression vector for TGF-beta 1 as a means of regulating endogenous TGF-beta 1 expression in tumor cells. Antisense-transfected FET human colon carcinoma cells showed a fivefold reduction in TGF-beta 1 mRNA and 15-fold reduction in TGF-beta 1 secretion. Antisense mRNA was detected in transfected cells by an RNase protection assay. Compared to control cells, cultured antisense-transfected cells showed a reduction in lag phase time rather than a change in doubling time. Cloning efficiencies of transfected cells were four times greater than control cells in anchorage-independent assays. Control cells did not form tumors at 5 x 10(5) in athymic nude mice. Antisense-transfected cells formed tumors in 40% of animals injected. At higher inocula (1 x 10(6) cells) antisense-transfected cells formed tumors in 100% of animals injected, but control cells still failed to form tumors. These results show that TGF-beta 1 acts as a negative growth regulator of human colon carcinoma cells in vivo as well as in vitro. Acquisition of partial or full resistance to such inhibitory effects may therefore contribute to tumor development and progression.

scholarly journals Bovine Herpesvirus 1 UL3.5 Interacts with Bovine Herpesvirus 1 α-Transinducing Factor

2000 ◽  
Vol 74 (6) ◽  
pp. 2876-2884 ◽  
Author(s):  
Ngan Lam ◽  
Geoffrey J. Letchworth
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bovine Herpesvirus ◽  
Mass Spectrometry Analysis ◽  
Bovine Herpesvirus 1 ◽  
Subcellular Compartment ◽  
Transfected Cells ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACT The bovine herpesvirus 1 (BHV-1) UL3.5 gene encodes a 126-amino-acid tegument protein. Homologs of UL3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking UL3.5 is deficient in viral egress but can be complemented by BHV-1 UL3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886–8892, 1997). The function of BHV-1 UL3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 UL3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 α-transinducing factor (αBTIF) as a BHV-1 UL3.5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, UL3.5 and αBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of UL3.5, but not 40 amino acids from the C terminus, abolished the UL3.5-αBTIF interaction both in vitro and in vivo. The interaction between UL3.5 and αBTIF may be important for BHV-1 maturation and regulation of αBTIF transactivation activity.

The Role of AKT and ERK in Regulating D-Cyclin Translation Inhibition and G1 Arrest in Multiple Myeloma Cells Treated with mTOR Inhibitors: Rationale for Combination Thereapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4795-4795
Author(s):  
Patrick J. Frost ◽  
YiJiang Shi ◽  
Carolyne Bardalaban ◽  
Bao Hoang ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Mtor Inhibitors ◽  
G1 Arrest ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Myeloma Cells ◽  
Transfected Cells ◽  
P38 Inhibitor

Abstract In a previous study, we showed that heightened AKT activity sensitized multiple myeloma (MM) cells to the in vivo anti-tumor effects of CCI-779. To test the mechanism of AKT’s regulatory role, we studied isogenic U266 MM cell lines transfected with an activated AKT allele or empty vector. The AKT-transfected cells were markedly more sensitive to cytostasis induced in vitro by rapamycin or in vivo by CCI-779. In contrast, cells with quiescent AKT were completely resistant. The ability of rapamycin and CCI-779 to inhibit D-cyclin expression was also significantly greater in AKT-transfected MM cells and this was, in part, due to a greater ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. As ERK/p38 activity can facilitate IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of rapamycin sensitivity. AKT-transfected cells demonstrated significantly decreased ERK and p38 activity, suggesting their involvement. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells while a p38 inhibitor had no effect. Furthermore, the combination of rapamycin and the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. These data support a scenario where ERK facilitates D-cyclin IRES function and heightened AKT activity down regulates this ERK-dependent phenomenon. Thus ERK and AKT activity are potential predictors of responsiveness to mTOR inhibitors.

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