scholarly journals Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 852
Author(s):  
Viacheslav V. Senichkin ◽  
Evgeniia A. Prokhorova ◽  
Boris Zhivotovsky ◽  
Gelina S. Kopeina
Keyword(s):  
Subcellular Fractionation ◽  
Nuclear Fraction ◽  
Apoptotic Cells ◽  
Apoptotic Bodies ◽  
Functional Studies ◽  
Ionic Detergents ◽  
Protein Functions ◽  
Indispensable Tool ◽  
Cell Fragments

Subcellular fractionation approaches remain an indispensable tool among a large number of biochemical methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochemical techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the standard protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the separation of nuclear and cytoplasmic contents. The described methodology is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insoluble subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.

scholarly journals A Trp-BODIPY cyclic peptide for fluorescence labelling of apoptotic bodies

2017 ◽  
Vol 53 (5) ◽  
pp. 945-948 ◽  
Author(s):  
Ramon Subiros-Funosas ◽  
Lorena Mendive-Tapia ◽  
Jesus Sot ◽  
John D. Pound ◽  
Nicole Barth ◽  
...  
Keyword(s):  
Cyclic Peptide ◽  
Synthesis And Characterization ◽  
Apoptotic Cells ◽  
Apoptotic Bodies ◽  
Fluorescence Labelling ◽  
Fluorogenic Peptide

We describe the synthesis and characterization of a Trp-BODIPY fluorogenic peptide for labelling subcellular bodies released by human apoptotic cells.

scholarly journals Characterization of adenylyl cyclase isoforms in rat peripheral pulmonary arteries

2001 ◽  
Vol 280 (6) ◽  
pp. L1359-L1369 ◽  
Author(s):  
Karen B. Jourdan ◽  
Nicola A. Mason ◽  
Lu Long ◽  
Peter G. Philips ◽  
Martin R. Wilkins ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Adenylyl Cyclase ◽  
Pulmonary Arteries ◽  
Bronchial Epithelium ◽  
Rt Pcr ◽  
Bronchial Smooth Muscle ◽  
Functional Studies ◽  
Kinase C ◽  
Rat Lungs

Activation of adenylyl cyclase (AC), of which there are 10 diversely regulated isoforms, is important in regulating pulmonary vascular tone and remodeling. Immunohistochemistry in rat lungs demonstrated that AC2, AC3, and AC5/6 predominated in vascular and bronchial smooth muscle. Isoforms 1, 4, 7, and 8 localized to the bronchial epithelium. Exposure of animals to hypoxia did not change the pattern of isoform expression. RT-PCR confirmed mRNA expression of AC2, AC3, AC5, and AC6 and demonstrated AC7 and AC8 transcripts in smooth muscle. Western blotting confirmed the presence of AC2, AC3, and AC5/6 proteins. Functional studies provided evidence of cAMP regulation by Ca2+ and protein kinase C-activated but not Gi-inhibited pathways, supporting a role for AC2 and a Ca2+-stimulated isoform, AC8. However, NKH-477, an AC5-selective activator, was more potent than forskolin in elevating cAMP and inhibiting serum-stimulated [3H]thymidine incorporation, supporting the presence of AC5. These studies demonstrate differential expression of AC isoforms in rat lungs and provide evidence that AC2, AC5, and AC8 are functionally important in cAMP regulation and growth pathways in pulmonary artery myocytes.

Characterization of Different Deoxyribonucleases in Human Lymphocytes

1975 ◽  
Vol 30 (11-12) ◽  
pp. 781-784 ◽  
Author(s):  
E. Jürgen Zöllner ◽  
Hans Störger ◽  
Hans-Joachim Breter ◽  
Rudolf Zahn
Keyword(s):  
Human Lymphocytes ◽  
Disc Electrophoresis ◽  
The Other ◽  
Lower Activity ◽  
Nuclear Fraction ◽  
Polyacrylamide Gels ◽  
Cytoplasmic Fraction ◽  
Acid Deoxyribonuclease ◽  
Deoxyribonuclease Activity

Abstract Deoxyribonucleases, Disc Electrophoresis, Lymphocytes Four groups of deoxyribonuclease activities from human lymphocytes have been characterized by deoxyribonuclease assay in DNA-containing polyacrylamide gels following their separation by disc-electrophoresis. All activities hydrolyse DNA endonucleolytically. One neutral deoxyribo­ nuclease found in the cytoplasmic fraction prefers native or UV-irradiated DNA over denatured DNA as substrate and is a 5′-monoester former. Two groups of acid deoxyribonuclease activities are detectable in the nuclear fraction. Both are 3′-monoester formers. One is as well active with denatured DNA as with native DNA, the other one shows the same activity with native and UV-irradiated DNA but lower activity with denatured DNA. An alkaline deoxyribonuclease activity, also localized in the nucleus, is a 5′ -monoester DNA as substrate.

scholarly journals Deletion of small ankyrin 1 (sAnk1) isoforms results in structural and functional alterations in aging skeletal muscle fibers

2015 ◽  
Vol 308 (2) ◽  
pp. C123-C138 ◽  
Author(s):  
E. Giacomello ◽  
M. Quarta ◽  
C. Paolini ◽  
R. Squecco ◽  
P. Fusco ◽  
...  
Keyword(s):  
Structural Damage ◽  
Skeletal Muscles ◽  
Functional Characterization ◽  
Sr Proteins ◽  
Endurance Test ◽  
Tubular Aggregates ◽  
Functional Studies ◽  
Old Mice ◽  
Contractile Performance

Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4- to 6-mo-old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4–6 mo of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12–15 mo of age) extensor digitorum longus (EDL) skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared with control mice. Moreover, reduced Qγcharge and L-type Ca2+current, which are indexes of affected excitation-contraction coupling, were observed in diaphragm fibers from 12- to 15-mo-old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.

Abstract 23: Characterization of MitoTimer, a Novel Tool for Monitoring Mitochondrial Turnover

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Christine A Thornton ◽  
Allen M Andres ◽  
Genaro Hernandez ◽  
Jon Sin ◽  
Roberta Gottlieb
Keyword(s):  
Protein Import ◽  
Spatial Information ◽  
Fluorescent Protein ◽  
Mitochondrial Dynamics ◽  
Inducible Promoter ◽  
Subcellular Fractionation ◽  
Fluorescent Reporter ◽  
Hek 293 ◽  
Mitochondrial Turnover

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, developed by Terskikh and colleagues. Its fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial targeting sequence (coined “MitoTimer”) and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and induced production with doxycycline. Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Doxycycline addition for as little as 1hr was sufficient to label mitochondria. MitoTimer was detected as early as 4hr following doxycycline addition, and persisted in mitochondria for at least 72hr. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. MitoTimer matured to red fluorescence within 48hr, at which time a second pulse of doxycycline induced expression of green (immature) MitoTimer which was selectively incorporated into a subset of mitochondria actively engaged in protein import. The extent of new protein incorporation during a second pulse was increased under conditions of mito-biogenesis and reduced if mitochondrial membrane potential was dissipated. We conclude that MitoTimer can be used to monitor mitophagy and biogenesis.

Characterization of nuclear structures containing superhelical DNA

1976 ◽  
Vol 22 (2) ◽  
pp. 303-324 ◽  
Author(s):  
P.R. Cook ◽  
I.A. Brazell ◽  
E. Jost
Keyword(s):  
Protein Content ◽  
Ionic Detergents ◽  
High Concentrations ◽  
Nuclear Structures ◽  
Intercalating Agents ◽  
Made In

Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated.

scholarly journals Non3 is an essential Drosophila gene required for proper nucleolus assembly

2019 ◽  
Vol 23 (2) ◽  
pp. 190-198
Author(s):  
E. N. Andreyeva ◽  
A. A. Ogienko ◽  
A. A. Yushkova ◽  
J. V. Popova ◽  
G. A. Pavlova ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Large Deletion ◽  
Protein Product ◽  
P Element ◽  
Post Translational Modification ◽  
Allelic Series ◽  
Larval Brain ◽  
Functional Studies ◽  
Mild Reduction

The nucleolus is a dynamic non-membrane-bound nuclear organelle, which plays key roles not only in ribosome biogenesis but also in many other cellular processes. Consistent with its multiple functions, the nucleolus has been implicated in many human diseases, including cancer and degenerative pathologies of the nervous system and heart. Here, we report the characterization of the Drosophila Non3 (Novel nucleolar protein 3) gene, which encodes a protein homologous to the human Brix domain-containing Rpf2 that has been shown to control ribosomal RNA (rRNA) processing. We used imprecise P-element excision to generate four new mutant alleles in the Non3 gene. Complementation and phenotypic analyses showed that these Non3 mutations can be arranged in an allelic series that includes both viable and lethal alleles. The strongest lethal allele (Non3∆600) is a genetically null allele that carries a large deletion of the gene and exhibits early lethality when homozygous. Flies heterozygous for Non3∆600 occasionally exhibit a mild reduction in the bristle size, but develop normally and are fertile. However, heteroallelic combinations of viable Non3 mutations (Non3197, Non3310 and Non3259) display a Minute-like phenotype, consisting in delayed development and short and thin bristles, suggesting that they are defective in ribosome biogenesis. We also demonstrate that the Non3 protein localizes to the nucleolus of larval brain cells and it is required for proper nucleolar localization of Fibrillarin, a protein important for post-translational modification and processing of rRNAs. In summary, we generated a number of genetic and biochemical tools that were exploited for an initial characterization of Non3, and will be instrumental for future functional studies on this gene and its protein product.

scholarly journals Characterization of MGC2, a Mycoplasma gallisepticum Cytadhesin with Homology to the Mycoplasma pneumoniae 30-Kilodalton Protein P30 and Mycoplasma genitalium P32

1998 ◽  
Vol 66 (7) ◽  
pp. 3436-3442 ◽  
Author(s):  
Linda L. Hnatow ◽  
Calvin L. Keeler ◽  
Laura L. Tessmer ◽  
Kirk Czymmek ◽  
John E. Dohms
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Reverse Transcription ◽  
Mycoplasma Genitalium ◽  
Mycoplasma Gallisepticum ◽  
Immunogold Labeling ◽  
Respiratory Pathogen ◽  
Functional Studies ◽  
Reverse Transcription Pcr ◽  
Attachment Inhibition

ABSTRACT A second cytadhesin-like protein, MGC2, was identified in the avian respiratory pathogen Mycoplasma gallisepticum. The 912-nucleotide mgc2 gene encodes a 32.6-kDa protein with 40.9 and 31.4% identity with the M. pneumoniae P30 andM. genitalium P32 cytadhesins, respectively. Functional studies with reverse transcription-PCR, immunoblotting, double-sided immunogold labeling, and attachment inhibition assays demonstrated homology to the human mycoplasmal P30 and P32 cytadhesins. These findings suggest that there is a family of cytadhesin genes conserved among pathogenic mycoplasmas infecting widely divergent hosts.

scholarly journals Genome-Wide Identification and Characterization of theLRR-RLKGene Family in TwoVerniciaSpecies

2015 ◽  
Vol 2015 ◽  
pp. 1-17 ◽  
Author(s):  
Huiping Zhu ◽  
Yangdong Wang ◽  
Hengfu Yin ◽  
Ming Gao ◽  
Qiyan Zhang ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Consensus Sequence ◽  
Expression Patterns ◽  
Functional Studies ◽  
Industrial Oil ◽  
Extensive Evaluation ◽  
Genome Wide ◽  
Pathogen Response ◽  
Lrr Domain

Leucine-rich repeat receptor-like kinases (LRR-RLKs) make up the largest group of RLKs in plants and play important roles in many key biological processes such as pathogen response and signal transduction. To date, most studies on LRR-RLKs have been conducted on model plants. Here, we identified 236 and 230LRR-RLKsin two industrial oil-producing trees:Vernicia fordiiandVernicia montana, respectively. Sequence alignment analyses showed that the homology of the RLK domain (23.81%) was greater than that of the LRR domain (9.51%) among theVf/VmLRR-RLKs. The conserved motif of the LRR domain inVf/VmLRR-RLKsmatched well the known plant LRR consensus sequence but differed at the third last amino acid (W or L). Phylogenetic analysis revealed thatVf/VmLRR-RLKswere grouped into 16 subclades. We characterized the expression profiles ofVf/VmLRR-RLKsin various tissue types including root, leaf, petal, and kernel. Further investigation revealed thatVf/VmLRR-RLKorthologous genes mainly showed similar expression patterns in response to tree wilt disease, except 4 pairs ofVf/VmLRR-RLKsthat showed opposite expression trends. These results represent an extensive evaluation ofLRR-RLKsin two industrial oil trees and will be useful for further functional studies on these proteins.

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