Rapid purification of biologically active individual histone messenger RNAs by hybridization to cloned DNA linked to cellulose

Biochemistry ◽  
1979 ◽  
Vol 18 (1) ◽  
pp. 208-213 ◽  
Author(s):  
Geoffrey Childs ◽  
Shoshana Levy ◽  
Laurence H. Kedes
Keyword(s):  
Biologically Active ◽  
Messenger Rnas ◽  
Active Individual ◽  
Rapid Purification

scholarly journals Significantly Improved Recovery of Recombinant Sonchus Yellow Net Rhabdovirus by Expressing the Negative-Strand Genomic RNA

Viruses ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1459
Author(s):  
Xiaonan Ma ◽  
Zhenghe Li
Keyword(s):  
Matrix Protein ◽  
Plant Viruses ◽  
Biologically Active ◽  
Messenger Rnas ◽  
Genomic Rna ◽  
Genomic Rnas ◽  
Double Stranded Rna ◽  
Core Proteins ◽  
Negative Sense

Generation of recombinant negative-stranded RNA viruses (NSVs) from plasmids involves in vivo reconstitution of biologically active nucleocapsids and faces a unique antisense problem where the negative-sense viral genomic RNAs can hybridize to viral messenger RNAs. To overcome this problem, a positive-sense RNA approach has been devised through expression of viral antigenomic (ag)RNA and core proteins for assembly of antigenomic nucleocapsids. Although this detour strategy works for many NSVs, the process is still inefficient. Using Sonchus yellow net rhabdovirus (SYNV) as a model; here, we develop a negative-sense genomic RNA-based approach that increased rescue efficiency by two orders of magnitude compared to the conventional agRNA approach. The system relied on suppression of double-stranded RNA induced antiviral responses by co-expression of plant viruses-encoded RNA silencing suppressors or animal viruses-encoded double-stranded RNA antagonists. With the improved approach, we were able to recover a highly attenuated SYNV mutant with a deletion in the matrix protein gene which otherwise could not be rescued via the agRNA approach. Reverse genetics analyses of the generated mutant virus provided insights into SYNV virion assembly and morphogenesis. This approach may potentially be applicable to other NSVs of plants or animals.

Fractionation of biologically active messenger RNAs by HPLC gel filtration

1982 ◽  
Vol 107 (4) ◽  
pp. 1559-1565 ◽  
Author(s):  
Lutz Graeve ◽  
Wolfgang Goemann ◽  
Peter Földi ◽  
Joachim Kruppa
Keyword(s):  
Gel Filtration ◽  
Biologically Active ◽  
Messenger Rnas

scholarly journals Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs

2021 ◽  
Vol 118 (40) ◽  
pp. e2018899118
Author(s):  
Divya Ram Jayaram ◽  
Sigal Frost ◽  
Chanan Argov ◽  
Vijayasteltar Belsamma Liju ◽  
Nikhil Ponnoor Anto ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Kinase Inhibitor ◽  
Open Reading Frames ◽  
Biologically Active ◽  
Messenger Rnas ◽  
Active Peptides ◽  
Protein Levels ◽  
Lower Protein ◽  
Cancer Tumor ◽  
Upstream Open Reading Frames

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5′ untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

scholarly journals Stem Cell-Derived Nanovesicles: A Novel Cell-Free Therapy for Wound Healing

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Jianghong Huang ◽  
Jun Zhang ◽  
Jianyi Xiong ◽  
Shuqing Sun ◽  
Jiang Xia ◽  
...  
Keyword(s):  
Wound Healing ◽  
Stem Cell ◽  
Wound Repair ◽  
Current Knowledge ◽  
Healing Process ◽  
Biologically Active ◽  
Research Progress ◽  
Wound Healing Process ◽  
Messenger Rnas ◽  
Skin Wound Healing

Wound healing and regeneration are a dynamic and complex process that requires a collaborative effort between growth factors, epidermal cells, dermal cells, extracellular matrix, and vessels local to the wound area. Mesenchymal stem cells participate in the recruitment site, mainly by releasing secretory factors and matrix proteins to promote wound healing. Stem cell-derived nanovesicles (CDNs), including microvesicles, exosomes, and exosome mimetics, contain most of the biologically active substances of their parent cells and have similar effects. CDNs can shuttle various proteins, messenger RNAs, and microRNAs to regulate the activity of receptor cells, and they play important roles in skin wound healing. This article reviews recent research progress on CDNs for wound repair. We summarize current knowledge on how CDNs regulate immunity, fibroblast activity, angiogenesis, and scar formation in the wound healing process. This review can help researchers explore new treatment strategies to enhance the therapeutic efficacy of CDNs, which have a promising future as naturally cell-free therapies.

scholarly journals A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule

2011 ◽  
Vol 9 (5) ◽  
pp. 618-628 ◽  
Author(s):  
Martin F. Gisby ◽  
Philip Mellors ◽  
Panagiotis Madesis ◽  
Marianne Ellin ◽  
Hugh Laverty ◽  
...  
Keyword(s):  
Biologically Active ◽  
Synthetic Gene ◽  
Active Molecule ◽  
Rapid Purification

scholarly journals Biological activity of mesenchymal stem cells secretome as a basis for cell-free therapeutic approach

2020 ◽  
Vol 6 (1) ◽  
pp. 57-68 ◽  
Author(s):  
Liubov A. Pokrovskaya ◽  
Ekaterina V. Zubareva ◽  
Sergey V. Nadezhdin ◽  
Anna S. Lysenko ◽  
Tatyana L. Litovkina
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stromal Cells ◽  
Adult Stem Cells ◽  
Neuroprotective Effect ◽  
Biologically Active ◽  
Adipose Derived Stem Cells ◽  
Messenger Rnas ◽  
Micro Rnas ◽  
Non Coding Rnas

Mesenchymal stem (stromal) cells (MSCs) are self-renewing, cultured adult stem cells which secrete a complex set of multiple soluble biologically active molecules such as chemokines, and cytokines, cell adhesion molecules, lipid mediators, interleukins (IL), growth factors (GFs), hormones, micro RNAs (miRNAs), long non-coding RNAs (lncRNAs), messenger RNAs (mRNAs), exosomes, as well as microvesicles, the secretome. MSCs of various origin, including adipose-derived stem cells (ASCs), bone marrow derived mesenchymal stem cells (BM-MSCs), human uterine cervical stem cells (hUCESCs), may be good candidates for obtaining secretome-derived products. Different population of MSCs can secret different factors which could have anti-inflammatory, anti-apoptotic, anti-fibrotic activities, a neuroprotective effect, could improve bone, muscle, liver regeneration and wound healing. Therefore, the paracrine activity of conditioned medium obtained when cultivating MSCs, due to a plethora of bioactive factors, was assumed to have the most prominent cell-free therapeutic impact and can serve as a better option in the field of regenerative medicine in future.

scholarly journals Rapid Purification of Endotoxin-Free RTX Toxins

Toxins ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 336 ◽  
Author(s):  
Ondrej Stanek ◽  
Jiri Masin ◽  
Radim Osicka ◽  
David Jurnecka ◽  
Adriana Osickova ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Simple Procedure ◽  
Biologically Active ◽  
Kingella Kingae ◽  
E Coli ◽  
Highly Active ◽  
Rtx Toxins ◽  
Rapid Purification ◽  
Triton X

Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).

Uultrastructure of Microtubules

1972 ◽  
Vol 30 ◽  
pp. 252-253
Author(s):  
Ariaki Nagayama
Keyword(s):  
Fine Structure ◽  
Cultured Cells ◽  
Present Report ◽  
Confluent Monolayer ◽  
Purification Method ◽  
Vinca Rosea ◽  
L Cells ◽  
Antimitotic Activity ◽  
Monolayer Cultures ◽  
Rapid Purification

Vinblastine(Vb) or vincristine, alkaloid derived from Vinca rosea is known for its antimitotic activity by regrouping of microtubules into paracrystalline form within the cells. A rapid purification method of vinblastine-induced microtubular paracrystals(PC) has provided us with a fresh and pure microtubular material demonstrating the presence of a labile ATPase associated with the PC. The present report is concerned with the fine structure of purified microtubules of mammalian cultured cells.Confluent monolayer cultures of L cells were incubated for 20hrs with 10-5 M Vb (donated from Shionogi Seiyaku & Co., Osaka, Japan).

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