Microtubules: Inhibition of Spontaneous in vitro Assembly by Non-neural Cell Extracts

1976 ◽  
pp. 161-174 ◽  
Author(s):  
J. Bryan ◽  
B. W. Nagle
Keyword(s):  
Neural Cell ◽  
Cell Extracts ◽  
In Vitro Assembly

scholarly journals In Vitro Assembly of Human H/ACA Small Nucleolar RNPs Reveals Unique Features of U17 and Telomerase RNAs

2000 ◽  
Vol 20 (9) ◽  
pp. 3037-3048 ◽  
Author(s):  
François Dragon ◽  
Vanda Pogačić ◽  
Witold Filipowicz
Keyword(s):  
Full Length ◽  
Stem Loop ◽  
Rna Transcripts ◽  
Cell Extracts ◽  
In Vitro Assembly ◽  
Tail Structure ◽  
Human Telomerase ◽  
Nucleolar Rnas

ABSTRACT The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs. They usually fold into a two-domain hairpin-hinge-hairpin-tail structure, with the conserved motifs H and ACA located in the hinge and tail, respectively. Synthetic RNA transcripts and extracts from HeLa cells were used to reconstitute human U17 and other H/ACA ribonucleoproteins (RNPs) in vitro. Competition and UV cross-linking experiments showed that proteins of about 60, 29, 23, and 14 kDa interact specifically with U17 RNA. Except for U17, RNPs could be reconstituted only with full-length H/ACA snoRNAs. For U17, the 3′-terminal stem-loop followed by box ACA (U17/3′st) was sufficient to form an RNP, and U17/3′st could compete other full-length H/ACA snoRNAs for assembly. The H/ACA-like domain that constitutes the 3′ moiety of human telomerase RNA (hTR), and its 3′-terminal stem-loop (hTR/3′st), also could form an RNP by binding H/ACA proteins. Hence, the 3′-terminal stem-loops of U17 and hTR have some specific features that distinguish them from other H/ACA RNAs. Antibodies that specifically recognize the human GAR1 (hGAR1) protein could immunoprecipitate H/ACA snoRNAs and hTR from HeLa cell extracts, which demonstrates that hGAR1 is a component of H/ACA snoRNPs and telomerase in vivo. Moreover, we show that in vitro-reconstituted RNPs contain hGAR1 and that binding of hGAR1 does not appear to be a prerequisite for the assembly of the other H/ACA proteins.

In vitro assembly of poliovirus 14 S subunits: Identification of the assembly promoting activity of infected cell extracts

Virology ◽  
1991 ◽  
Vol 180 (2) ◽  
pp. 781-787 ◽  
Author(s):  
B. Rombaut ◽  
A. Foriers ◽  
A. Boeyé
Keyword(s):  
Infected Cell ◽  
Cell Extracts ◽  
In Vitro Assembly

scholarly journals Two Inactive Fragments of the Integral RNA Cooperate To Assemble Active Telomerase with the Human Protein Catalytic Subunit (hTERT) In Vitro

1999 ◽  
Vol 19 (9) ◽  
pp. 6207-6216 ◽  
Author(s):  
Valerie M. Tesmer ◽  
Lance P. Ford ◽  
Shawn E. Holt ◽  
Bryan C. Frank ◽  
Xiaoming Yi ◽  
...  
Keyword(s):  
De Novo ◽  
Telomerase Activity ◽  
Catalytic Subunit ◽  
Human Protein ◽  
Cell Extracts ◽  
In Vitro Assembly ◽  
Reticulocyte Lysates ◽  
Minimal Sequence ◽  
Human Telomerase

ABSTRACT We have mapped the 5′ and 3′ boundaries of the region of the human telomerase RNA (hTR) that is required to produce activity with the human protein catalytic subunit (hTERT) by using in vitro assembly systems derived from rabbit reticulocyte lysates and human cell extracts. The region spanning nucleotides +33 to +325 of the 451-base hTR is the minimal sequence required to produce levels of telomerase activity that are comparable with that made with full-length hTR. Our results suggest that the sequence approximately 270 bases downstream of the template is required for efficient assembly of active telomerase in vitro; this sequence encompasses a substantially larger portion of the 3′ end of hTR than previously thought necessary. In addition, we identified two fragments of hTR (nucleotides +33 to +147 and +164 to +325) that cannot produce telomerase activity when combined separately with hTERT but can function together to assemble active telomerase. These results suggest that the minimal sequence of hTR can be divided into two sections, both of which are required for de novo assembly of active telomerase in vitro.

In vitro assembly of 2-D crystals from partially purified EA1 protein secreted by Bacillus anthracis strain Delta Sterne-1

1994 ◽  
Vol 52 ◽  
pp. 276-277
Author(s):  
Mary Beth Downs ◽  
Wilson Ribot ◽  
Joseph W. Farchaus
Keyword(s):  
Cell Wall ◽  
Molecular Sieves ◽  
Cell Envelope ◽  
Single Species ◽  
Supporting Cell ◽  
Surface Layers ◽  
Rabbit Igg ◽  
In Vitro Assembly ◽  
Covalently Linked

Many bacteria possess surface layers (S-layers) that consist of a two-dimensional protein lattice external to the cell envelope. These S-layer arrays are usually composed of a single species of protein or glycoprotein and are not covalently linked to the underlying cell wall. When removed from the cell, S-layer proteins often reassemble into a lattice identical to that found on the cell, even without supporting cell wall fragments. S-layers exist at the interface between the cell and its environment and probably serve as molecular sieves that exclude destructive macromolecules while allowing passage of small nutrients and secreted proteins. Some S-layers are refractory to ingestion by macrophages and, generally, bacteria are more virulent when S-layers are present.When grown in rich medium under aerobic conditions, B. anthracis strain Delta Sterne-1 secretes large amounts of a proteinaceous extractable antigen 1 (EA1) into the growth medium. Immunocytochemistry with rabbit polyclonal anti-EAl antibody made against the secreted protein and gold-conjugated goat anti-rabbit IgG showed that EAI was localized at the cell surface (fig 1), which suggests its role as an S-layer protein.

scholarly journals Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties

2021 ◽  
Vol 22 (3) ◽  
pp. 1124
Author(s):  
Mafalda Giovanna Reccia ◽  
Floriana Volpicelli ◽  
Eirkiur Benedikz ◽  
Åsa Fex Svenningsen ◽  
Luca Colucci-D’Amato
Keyword(s):  
Cell Line ◽  
Low Cost ◽  
Neural Cell ◽  
New Drugs ◽  
High Yield ◽  
Time Saving ◽  
Interacting Protein ◽  
Neuronal Marker ◽  
Differentiation Status

Neural stem cells represent a powerful tool to study molecules involved in pathophysiology of Nervous System and to discover new drugs. Although they can be cultured and expanded in vitro as a primary culture, their use is hampered by their heterogeneity and by the cost and time needed for their preparation. Here we report that mes-c-myc A1 cells (A1), a neural cell line, is endowed with staminal properties. Undifferentiated/proliferating and differentiated/non-proliferating A1 cells are able to generate neurospheres (Ns) in which gene expression parallels the original differentiation status. In fact, Ns derived from undifferentiated A1 cells express higher levels of Nestin, Kruppel-like factor 4 (Klf4) and glial fibrillary protein (GFAP), markers of stemness, while those obtained from differentiated A1 cells show higher levels of the neuronal marker beta III tubulin. Interestingly, Ns differentiation, by Epidermal Growth Factors (EGF) and Fibroblast Growth Factor 2 (bFGF) withdrawal, generates oligodendrocytes at high-yield as shown by the expression of markers, Galactosylceramidase (Gal-C) Neuron-Glial antigen 2 (NG2), Receptor-Interacting Protein (RIP) and Myelin Basic Protein (MBP). Finally, upon co-culture, Ns-A1-derived oligodendrocytes cause a redistribution of contactin-associated protein (Caspr/paranodin) protein on neuronal cells, as primary oligodendrocytes cultures, suggesting that they are able to form compact myelin. Thus, Ns-A1-derived oligodendrocytes may represent a time-saving and low-cost tool to study the pathophysiology of oligodendrocytes and to test new drugs.

scholarly journals Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Graziana Esposito ◽  
Bijorn Omar Balzamino ◽  
Egidio Stigliano ◽  
Filippo Biamonte ◽  
Andrea Urbani ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Cell Signalling ◽  
Specific Protein ◽  
Conditioned Media ◽  
Cell Extracts ◽  
Dose Dependent Decrease ◽  
Significant Expression ◽  
Biomolecular Analysis ◽  
Protein Signature

AbstractWe previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines’ signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1–100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors’ activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.

scholarly journals In Vitro Assembly of l-Asparaginase Subunits

1974 ◽  
Vol 249 (13) ◽  
pp. 4175-4180 ◽  
Author(s):  
Sidney Shifrin ◽  
Catherine L. Parrott
Keyword(s):  
In Vitro Assembly

scholarly journals A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

2014 ◽  
Vol 88 (6) ◽  
pp. 3577-3585 ◽  
Author(s):  
J. B. Munro ◽  
A. Nath ◽  
M. Farber ◽  
S. A. K. Datta ◽  
A. Rein ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Virus Like Particles ◽  
In Vitro Assembly ◽  
Hiv 1
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