Cytosolic Delivery of Argininosuccinate Synthetase Using a Cell-Permeant Miniature Protein

Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non-toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non-covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ-PEG) and a pyrrolidone containing linear derivative (PZ-PYR) for their ability to intracellularly deliver FITC-avidin, a model protein. In endothelial cells, PZ-PYR/protein exhibited both faster internalization and higher uptake levels than PZ-PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ-PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ-PEG/avidin colocalized more profusely with endo-lysosomes, and PZ-PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ-PYR-delivered anti-F-actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell-impermeable Bax-BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ-PYR, demonstrating endo-lysosomal escape. These biodegradable PZs were non-toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

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Supramolecular caging for cytosolic delivery of anionic probes

Chemical Science ◽

10.1039/c9sc02906k ◽

2019 ◽

Vol 10 (39) ◽

pp. 8930-8938 ◽

Cited By ~ 4

Author(s):

Héctor Fernández-Caro ◽

Irene Lostalé-Seijo ◽

Miguel Martínez-Calvo ◽

Jesús Mosquera ◽

José L. Mascareñas ◽

...

Keyword(s):

Alexa Fluor ◽

Cytosolic Delivery ◽

Anionic Charge ◽

Cell Permeable Peptide ◽

A Cell

A cell-permeable peptide-cage hybrid allows the cytosolic delivery of cage-interacting probes, including pyranine, carboxyfluorescein, and Alexa Fluor dyes, which are usually membrane-impermeable due to their high anionic charge.

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Intracellular delivery of a native functional protein using cell-penetrating peptide functionalized cubic MSNs with ultra-large mesopores

Journal of Materials Chemistry B ◽

10.1039/c8tb00330k ◽

2018 ◽

Vol 6 (21) ◽

pp. 3456-3465 ◽

Cited By ~ 2

Author(s):

Sang-Eun Bae ◽

Soo Kyung Lyu ◽

Ki-Jung Kim ◽

Hee Joo Shin ◽

Hyockman Kwon ◽

...

Keyword(s):

Click Reaction ◽

Intracellular Delivery ◽

Cell Penetrating Peptide ◽

Functional Protein ◽

Native Form ◽

Cytosolic Delivery ◽

Cell Penetrating ◽

A Cell ◽

Large Mesopores

Pore-enlarged 3D cubic cMSNs were successfully prepared, and their surfaces were functionalized by a cell-penetrating R8-peptide through the click reaction for cytosolic delivery of a functional protein in its native form.

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Effect of cyclic AMP and prostaglandin E2 on the induction of nitric oxide- and prostanoid-forming pathways in cultured rat mesangial cells

Biochemical Journal ◽

10.1042/bj3130617 ◽

1996 ◽

Vol 313 (2) ◽

pp. 617-623 ◽

Cited By ~ 46

Author(s):

Rolf M. NÜSING ◽

Thomas KLEIN ◽

Josef PFEILSCHIFTER ◽

Volker ULLRICH

Keyword(s):

Cyclic Amp ◽

Prostaglandin E2 ◽

Mesangial Cells ◽

Mechanistic Explanation ◽

No Synthase ◽

Argininosuccinate Synthetase ◽

Inducible No Synthase ◽

Low Concentrations ◽

Signalling Molecule ◽

A Cell

Cyclic AMP (cAMP) represents an important cellular signalling molecule. We analysed the effect of dibutyryl cAMP (db-cAMP), a cell-permeable and stable derivative of cAMP, on the regulation and expression of cyclo-oxygenase 2, inducible NO synthase and argininosuccinate synthetase. We observed different transcriptional regulation of these enzymes depending on the db-cAMP concentration used. Low concentrations of db-cAMP in the range 10-50 μM elevated levels of cyclo-oxygenase 2 mRNA, protein and activity, but not the respective mRNA and protein concentrations of the inducible NO synthase or argininosuccinate synthetase. At higher concentrations a massive induction of the latter two enzymes was also apparent. Expression of prostacyclin synthase and argininosuccinate lyase, secondary enzymes of NO-and prostanoid-forming pathways, was not stimulated by db-cAMP. Prostaglandin E2, known to be an intracellular physiological trigger of cAMP formation, stimulated only cyclo-oxygenase 2 expression and activity at a concentration of 10 μM, and not inducible NO synthase. The induction of the mRNA for the transcription factors JunB and p65, a component of the NFĸB complex, by prostaglandin treatment of the cells might be a possible mechanistic explanation for this observation.

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Effect of Niacin on Mitochondria of Allium Cepa Root Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060404 ◽

1967 ◽

Vol 25 ◽

pp. 78-79

Author(s):

M. Arif Hayat

Keyword(s):

Nicotinamide Adenine Dinucleotide ◽

Hydrogen Transfer ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Nicotinamide Adenine ◽

Root Tips ◽

Root Cells ◽

Transfer Processes ◽

Standard Procedures ◽

A Cell ◽

Critical Interest

Although it is recognized that niacin (pyridine-3-carboxylic acid), incorporated as the amide in nicotinamide adenine dinucleotide (NAD) or in nicotinamide adenine dinucleotide phosphate (NADP), is a cofactor in hydrogen transfer in numerous enzyme reactions in all organisms studied, virtually no information is available on the effect of this vitamin on a cell at the submicroscopic level. Since mitochondria act as sites for many hydrogen transfer processes, the possible response of mitochondria to niacin treatment is, therefore, of critical interest.Onion bulbs were placed on vials filled with double distilled water in the dark at 25°C. After two days the bulbs and newly developed root system were transferred to vials containing 0.1% niacin. Root tips were collected at ¼, ½, 1, 2, 4, and 8 hr. intervals after treatment. The tissues were fixed in glutaraldehyde-OsO4 as well as in 2% KMnO4 according to standard procedures. In both cases, the tissues were dehydrated in an acetone series and embedded in Reynolds' lead citrate for 3-10 minutes.

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Ultrastructure of melanocyte-keratinocyte interactions and pigment transfer in vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084120 ◽

1978 ◽

Vol 36 (2) ◽

pp. 650-651

Author(s):

Raul I. Garcia ◽

Evelyn A. Flynn ◽

George Szabo

Keyword(s):

Direct Injection ◽

Skin Pigmentation ◽

Freeze Fracture ◽

Pigment Granule ◽

Time Lapse ◽

Ultrastructural Level ◽

Lanthanum Tracer ◽

A Cell

Skin pigmentation in mammals involves the interaction of epidermal melanocytes and keratinocytes in the structural and functional unit known as the Epidermal Melanin Unit. Melanocytes(M) synthesize melanin within specialized membrane-bound organelles, the melanosome or pigment granule. These are subsequently transferred by way of M dendrites to keratinocytes(K) by a mechanism still to be clearly defined. Three different, though not necessarily mutually exclusive, mechanisms of melanosome transfer have been proposed: cytophagocytosis by K of M dendrite tips containing melanosomes, direct injection of melanosomes into the K cytoplasm through a cell-to-cell pore or communicating channel formed by localized fusion of M and K cell membranes, release of melanosomes into the extracellular space(ECS) by exocytosis followed by K uptake using conventional phagocytosis. Variability in methods of transfer has been noted both in vivo and in vitro and there is evidence in support of each transfer mechanism. We Have previously studied M-K interactions in vitro using time-lapse cinemicrography and in vivo at the ultrastructural level using lanthanum tracer and freeze-fracture.

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Dendritic cells of the human epidermis: immuno-electron microscopic studies of la antigen reactivity

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100084090 ◽

1978 ◽

Vol 36 (2) ◽

pp. 644-645

Author(s):

G. Rowden ◽

M. G. Lewis ◽

T. M. Phillips

Keyword(s):

Dendritic Cells ◽

Langerhans Cells ◽

Cell Types ◽

Cell Type ◽

Electron Microscopic ◽

La Antigen ◽

Microscopic Studies ◽

A Cell ◽

C3 Receptors ◽

Antigen Reactivity

Langerhans cells of mammalian stratified squamous epithelial have proven to be an enigma since their discovery in 1868. These dendritic suprabasal cells have been considered as related to melanocytes either as effete cells, or as post divisional products. Although grafting experiments seemed to demonstrate the independence of the cell types, much confusion still exists. The presence in the epidermis of a cell type with morphological features seemingly shared by melanocytes and Langerhans cells has been especially troublesome. This so called "indeterminate", or " -dendritic cell" lacks both Langerhans cells granules and melanosomes, yet it is clearly not a keratinocyte. Suggestions have been made that it is related to either Langerhans cells or melanocyte. Recent studies have unequivocally demonstrated that Langerhans cells are independent cells with immune function. They display Fc and C3 receptors on their surface as well as la (immune region associated) antigens.

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Presence of antibody in virus-infected brain cells of SSPE ferrets

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100077591 ◽

1983 ◽

Vol 41 ◽

pp. 804-805

Author(s):

Hannah R. Brown ◽

Tammy L. Donato ◽

Halldor Thormar

Keyword(s):

Measles Virus ◽

Plasma Cells ◽

Subacute Sclerosing Panencephalitis ◽

Protein A ◽

Neutralizing Antibody ◽

Brain Cells ◽

Antibody Titers ◽

A Cell ◽

The Central Nervous System ◽

The Brain

Measles virus specific immunoglobulin G (IgG) has been found in the brains of patients with subacute sclerosing panencephalitis (SSPE), a slowly progressing disease of the central nervous system (CNS) in children. IgG/albumin ratios indicate that the antibodies are synthesized within the CNS. Using the ferret as an animal model to study the disease, we have been attempting to localize the Ig's in the brains of animals inoculated with a cell associated strain of SSPE. In an earlier report, preliminary results using Protein A conjugated to horseradish peroxidase (PrAPx) (Dynatech Diagnostics Inc., South Windham, ME.) to detect antibodies revealed the presence of immunoglobulin mainly in antibody-producing plasma cells in inflammatory lesions and not in infected brain cells.In the present experiment we studied the brain of an SSPE ferret with neutralizing antibody titers of 1:1024 in serum and 1:512 in CSF at time of sacrifice 7 months after i.c. inoculation with SSPE measles virus-infected cells. The animal was perfused with saline and portions of the brain and spinal cord were immersed in periodate-lysine-paraformaldehyde (P-L-P) fixative. The ferret was not perfused with fixative because parts of the brain were used for virus isolation.

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Aberrant Type C Virus Production in a Cell Line Derived from Balb/3T3

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094693 ◽

1972 ◽

Vol 30 ◽

pp. 286-287

Author(s):

N. Savage ◽

A. Hackett

Keyword(s):

Electron Microscopy ◽

Cell Line ◽

Leukemia Virus ◽

Morphological Characteristics ◽

Virus Production ◽

Oncogenic Viruses ◽

Endogenous Virus ◽

Virus Particles ◽

Moloney Leukemia Virus ◽

A Cell

A cell line, UC1-B, which was derived from Balb/3T3 cells, maintains the same morphological characteristics of the non-transformed parental culture, and shows no evidence of spontaneous virus production. Survey by electron microscopy shows that the cell line consists of spindle-shaped cells with no unusual features and no endogenous virus particles.UC1-B cells respond to Moloney leukemia virus (MLV) infection by a change in morphology and growth pattern which is typical of cells transformed by sarcoma virus. Electron microscopy shows that the cells are now variable in shape (rounded, rhomboid, and spindle), and each cell type has some microvilli. Virtually all (90%) of the cells show virus particles developing at the cell surface and within the cytoplasm. Maturing viruses, typical of the oncogenic viruses, are found along with atypical tubular forms in the same cell.

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Head Size and DNA Content in Bacteriophage T4

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094255 ◽

1972 ◽

Vol 30 ◽

pp. 200-201

Author(s):

Fred Eiserling ◽

A. H. Doermann ◽

Linde Boehner

Keyword(s):

Electron Microscopy ◽

Dna Content ◽

Bacteriophage T4 ◽

Head Size ◽

Virus Particles ◽

Altered Protein ◽

A Cell ◽

Bacterial Viruses ◽

A Site ◽

Protein Shell

The control of form or shape inheritance can be approached by studying the morphogenesis of bacterial viruses. Shape variants of bacteriophage T4 with altered protein shell (capsid) size and nucleic acid (DNA) content have been found by electron microscopy, and a mutant (E920g in gene 66) controlling head size has been described. This mutant produces short-headed particles which contain 2/3 the normal DNA content and which are non-viable when only one particle infects a cell (Fig. 1).We report here the isolation of a new mutant (191c) which also appears to be in gene 66 but at a site distinct from E920g. The most striking phenotype of the mutant is the production of about 10% of the phage yield as “giant” virus particles, from 3 to 8 times longer than normal phage (Fig. 2).

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