38 results about "Cytoplasmic protein" patented technology

Contemplated compositions and methods are directed to the use of microvesicles from an optionally recombinant donor cell to impart a desirable effect to a recipient cell. In certain preferred aspects, RNA of the microvesicles is employed to achieve the desirable effect. For example, microvesicles are used in vitro to increase the number of passages of a cell growing in a medium, reduce serum and / or growth factor requirements of a cell growing in a medium, and / or delay differentiation of a cell growing in a medium. Further preferred aspects include use of the microvesicles as therapeutic agents in which RNA, a membrane protein, and / or a cytosolic protein encapsulated in or coupled to the microvesicle provide a therapeutic effect. Additionally, diagnostic methods are contemplated in which RNA of a microvesicle isolated from a mammal is associated with a condition of the mammal.

The invention provides methodologies and apparatus for producing devitalized soft-tissue implants where the implant retains metabolically non-viable and / or reproductively non-viable cells, and preferably retains large molecular weight cytoplasmic proteins, such implants produced both in small quantities and in commercializable quantities. Such soft-tissue implants include vascular graft substitutes. A devitalized graft is produced by subjecting the tissue sample to an induced pressure mediated flow of an extracting solution, optionally followed by inducing a pressure mediated flow of a salt solution, then washing the tissue to produce the devitalized graft. The devitalized grafts produced are uniform and non-immunogenic. The inventive method allows for the production of multiple devitalized soft tissue implants, where processing time is significantly less than prior art processes and the number of implants produced per day is increased over prior art processes. In clinical use, the devitalized grafts produced exhibit significantly improved in long-term durability and function, and enhanced recellularization post-implantation.

In one aspect, the invention provides a peptide comprising a chaperone-mediated autophagy (CMA)-targeting signal domain; a protein-binding domain that selectively binds to a target cytosolic protein; and a cell membrane penetrating domain (CMPD). In another aspect, the invention provides methods for reducing the intracellular expression level of an endogenous target protein in vitro and in an animal, wherein the method involves administration of the peptide. Methods are also provided for treating a pathological condition in an animal, the methods comprising administering the peptide to the animal. In one embodiment, the pathological condition is a neurodegenerative disease. In another embodiment of the invention, the target cytosolic protein is death associated protein kinase 1 and the CMPD is protein transduction domain of the HIV-1 Tat protein.

The invention discloses a transferring method for head cabbage cytoplasm male sterile line characterized by that, wild cabbage self-bred lines are utilized for 5-7 generation backcrossing with cabbage cytoplasmic protein sterility source. The advantages of the invention include stabilized male sterility, high sterility strain rate and sterility degree. The crossing rate of the obtained hybrid in strictly isolated area can be as high as 100%.

The present invention relates to cell membrane-derived nanovesicles, a method of preparing the same, and a pharmaceutical composition and a diagnostic kit using the nanovesicles. The cell membrane-derived nanovesicles according to the present invention may prevent the occurrence of potential side effects because intracellular materials (e.g., genetic materials and cytosolic proteins) unnecessary for delivery of therapeutic or diagnostic substances are removed from the nanovesicles. In addition, since the nanovesicles may be targeted to the specific types of cells or tissues, therapeutic or diagnostic substances may be predominantly delivered to the targeted cells or tissues while delivery of therapeutic or diagnostic substances to untargeted sites may be inhibited. Therefore, when the cell membrane-derived nanovesicles are applied to disease treatment, the side effects of therapeutic substances such as drugs may be reduced, so that suffering and inconvenience of patients may be alleviated during the course of treating diseases, and therapeutic efficacy may be improved. In addition, the cell membrane-derived nanovesicles of the present invention, in which substances for the treatment or diagnosis of diseases are loaded, and a method of preparing the nanovesicles may be used in vitro or in vivo for therapeutic or diagnostic purposes, or for experimental use.

The invention provides a method of making and secreting a non-secreted protein. The method includes expressing the protein from a nucleic acid construct which includes: (a) a mammary epithelial specific promoter; (b) a milk protein specific signal sequence which can direct the secretion of a protein; (c) optionally, a sequence which encodes a sufficient portion of the amino terminal coding region of a secreted protein to allow secretion in the milk of a transgenic mammal, of the non-secreted protein; and (d) a sequence which encodes a non-secreted protein, wherein elements (a), (b), optionally (c), and (d) are preferably operatively linked in the order recited. Both glutamic acid decarboxylase (GAD) and myelin basic protein (MBP), which are cytoplasmic proteins, have been produced by the methods of the present invention. The invention also provides methods for treating diabetes and multiple sclerosis using proteins produced by the methods of the present invention.

The invention discloses a method for extracting and detecting fish sperm membrane protein. The method comprises the following steps: pretreatment of a sample, fabrication of sperm suspension, suspension treatment of sperms, sperm membrane protein separation, sperm membrane protein degreasing purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) detection of sperm membrane protein and mass spectrometric detection of the sperm membrane protein. By adopting the method, the characteristics of a protease inhibitor and a TM-PEK (transmembrane-protein extraction kit) method are fitted on the basis of the traditional decontamination method, the cover degree of the extracted fish sperm membrane protein can be up to over 86%, and 0.6mg of membrane protein and 0.8mg of cytoplasmic protein can be obtained from 5*10<7> sperms on average. The basic data can be provided for verification of a fish sperm egg mechanism and a fertilization mechanism by the popularization and application of the method disclosed by the invention, guidance can be provided for culture and proliferation of fishes, especially reproductive breeding of migratory fishes in estuary sea, and a useful reference method is provided for research of sperm membrane protein of other aquatic species.

The invention provides a method for separating DNA (deoxyribonucleic acid) binding protein and accurately positioning DNA binding site, which comprises the following steps: DNA binding protein separation: carrying out PCR (polymerase chain reaction) amplification on a DNA segment to be researched by using biotin-labeled primers, sequentially adding nucleoprotein or cytoplast protein and avidin-coated magnetic beads, co-incubating, washing off nonspecific binding protein, and adding SDS (sodium dodecylsulfate) to denature and release the protein; and protein DNA binding site positioning: by using the DNA segment to be researched as a template, carrying out PCR reaction by using different primers to obtain overlapping DNA segments, and positioning the DNA binding site of the isolated protein according to the affinity difference of the overlapping DNA segments for the isolated protein.

The disclosure relates to a cytoplasmic protein complex comprising: (a) a first recombinant fusion protein comprising a kinase, fused to a first interaction polypeptide; and (b) a second recombinant fusion protein comprising a domain comprising a reporter phosphorylation site, whereby the domain is fused to a second interaction polypeptide. The disclosure relates further to a method to detect compound-compound-interaction using the cytoplasmic protein complex, and to cells comprising such cytoplasmic protein complex.

The invention relates to the technical field of biology, in particular to a marker, reagent or kit for detecting whether COVID-19 disease is cured. A plasma protein marker combination for diagnosing curing of a COVID-19 patient comprises a serine protease inhibitor 3, lymphocyte cytoplasmic protein 1, a peptidase inhibitor 16 and fetuin B. The results show that the area under the curve (AUC) valueof a receiver operating curve (ROC) of the four protein combinations predicting the cure of the COVID-19 patients is 0.947, the true positive rate of predicted cure is 82%, and the false positive rate is 18%. In addition, an enzyme-linked immunosorbent assay (ELISA) experiment finds that the protein concentration is significantly different in the plasma of the COVID-19 patient and a cured patient, which further confirms the accuracy of the combination for predicting the cure of the COVID-19 patient.

Disclosed herein are methods for quickly obtaining crude membranes from cells, including animal and plant cells. The methods include incubating cells in a buffer and forcing the cells through a filter that causes rupture of the cells, and then separating the resulting crude membranes from most cytosolic proteins.

The invention discloses a method for detecting entry of small molecule compounds into mitochondria at a cell in-situ level. The method particularly comprises the following steps: selecting suitable small molecule compounds to be incubated with cells; acquiring mitochondria with complete physiological structures without destroying cell mitochondria; collecting cytoplasm; incubating with the small molecule compounds and the acquired mitochondria; respectively pyrolyzing the obtained mitochondria after incubating the small molecule compounds and the cytoplasm collected before under non-denaturing conditions to acquire a mitochondrial protein lysate and a cytoplasmic protein lysate; and then, detecting whether the small molecule compounds enter the mitochondria at the cell in-situ level by combining with mass spectrum analysis. The method can be adopted to detect whether the small molecule compounds enter the mitochondria at the cell in-situ level, and can further detect a way in which the small molecule compounds enter the mitochondria, can be directly applied to target region verification of a pilot drug or screening of the pilot drug using the mitochondrion as an effector, and has the advantages of high reliability and high sensitivity.

The invention discloses a transhydrogenase-1 activity determination kit and a use method thereof. The kit comprises a mixed solution of a Tris-HCl buffer solution, sucrose and EDTA, a Tris-HCl buffersolution, NADH and AcPyADP. The method is based on the detection principle of replacing the NADP<+> with a synthetic substrate 3-acetylpyridine adenine dinucleotide phosphate (APADP<+>) and calculating TH-1 activity through measuring a light absorption increasing speed at 375nm of APADPH generated by the reduction of APADP<+> catalyzed by TH-1, can effectively eliminate the interference of NAD andhas high detection specificity. Through a simple work liquid, only through adding a sample and a work liquid into a microcuvette or a 96-well plate, the determination is finished and detection is convenient. The kit can effectively isolate cytoplasmic proteins and mitochondrial proteins and accurately determine TH-1 located in the mitochondrial inner membrane.

In one aspect, the invention provides a peptide comprising a chaperone-mediated autophagy (CMA)-targeting signal domain; a protein-binding domain that selectively binds to a target cytosolic protein; and a cell membrane penetrating domain (CMPD). In another aspect, the invention provides methods for reducing the intracellular expression level of an endogenous target protein in vitro and in an animal, wherein the method involves administration of the peptide. Methods are also provided for treating a pathological condition in an animal, the methods comprising administering the peptide to the animal. In one embodiment, the pathological condition is a neurodegenerative disease. In another embodiment of the invention, the target cytosolic protein is death associated protein kinase 1 and the CMPD is protein transduction domain of the HIV-1 Tat protein.

The invention discloses cytoplasmic protein-assisted enhanced anthocyanin-like fluorescent dye and a preparation method thereof. The main structure of the dye is shown in the description, wherein R1 and R2 are different substituent groups, and a value of n is 0 or 1. The dye provided by the invention has an excitation wavelength and emission wavelength of 550 nm or more and stable optical performance, and the fluorescence intensity value of the dye is significantly enhanced in the presence of a cytoplasmic protein; the dye and method provided by the invention have the following technical effects: the synthetic method of the dye is simple, raw materials are easy to obtain, the yield is high, and products are easy to separate and purify; at the same time, the structure of the dye is simple and stable, the dye is easily used for design of a probe, and detection of low-abundance targets in cells and living bodies by the probe can be effectively realized by utilizing the advantage that fluorescence of the dye is significantly enhanced in the presence of the cytoplasmic protein; and the dye has wide application prospects.

Transmembrane B ephrins and their Eph receptors signal bi-directionally. The presently claimed invention describes a cytoplasmic protein, designated PDZ-RGS3, which binds B ephrins through a PDZ domain, and has a regulator of heterotrimeric G protein signaling (RGS) domain. PDZ-RGS3 mediates signaling from the ephrin-B cytoplasmic tail. SDF-1, a chemokine with a G protein coupled receptor, or BDNF, act as chemoattractants for cerebellar granule cells, with SDF-1 action being selectively inhibited by soluble EphB receptor. The claimed invention reveals a pathway that links reverse signaling to cellular guidance, uncovers a novel mode of control for G proteins, and demonstrates a mechanism for selective regulation of responsiveness to neuronal guidance cues. Further, compositions and methods of use are provided for modulating cell migration as a function of chemokines and GPCR interaction, to aid in the treatment of disease states and medical conditions, including cancer and immune responses such as allergy and autoimmune responses. In one embodiment, a method of altering the sensitivity of a cell to a chemokine is provided using a PDZ-RGS3 protein.

The invention discloses a method for efficiently localizing exogenous mammal cytoplasmic protein in a cell nucleus and application thereof. According to the method, a pRK5-Sufu-GFP expression vector is digested by utilizing SalI single enzyme, then NLS is ligated into the vector by utilizing ligase, subsequently nuclear localization vectors pRK5-Sufu-nNLS-GFP (SnNG) with unequal numbers of inserted NLSs are obtained by transformation, and finally cytoplasmic protein is successfully localized in the cell nucleus. In the method disclosed by the invention, multiple NLSs can be inserted at one time, inserted n plasmid vectors (n being 1-5) are obtained simultaneously, and the exogenous cytoplasmic protein can be efficiently localized in the cell nucleus. The transfection of the plasmid into mouse fibroblasts (MEFs) is achieved by one-time construction of the cytoplasmic protein with 1-5 NLSs, so as to study the dose-effect relationship between the localization of the cytoplasmic protein incytoplasm / karyon and the biological activity and function of the cytoplasmic protein in cells.

The invention discloses a transferring method for head cabbage cytoplasm male sterile line characterized by that, wild cabbage self-bred lines are utilized for 5-7 generation backcrossing with cabbage cytoplasmic protein sterility source. The advantages of the invention include stabilized male sterility, high sterility strain rate and sterility degree. The crossing rate of the obtained hybrid in strictly isolated area can be as high as 100%.

The present invention relates to a novel bidentate motif that is composed of two adjacent residues of tyrosine and serine which have been found to be involved in the binding of crucial cytoplasmic proteins which are involved in cell signalling pathways. In some cases, the cytoplasmic proteins are ubiquitous proteins involved in cell signalling pathways that may include mitogenesis, transformation and survival. The bidentate motif may have a sequence alignment (SEQ ID NO: 71)N-X-X-Y- (X) 1-13-[R / K / H / Q]-[X / Ψ] 2-3-S / T-X-P;(SEQ ID NO: 72)Y- (X) 1-16-[R / K / H / Q]-[X / Ψ] 2-3-S / T-X-P;or(SEQ ID NO: 73)N-X-X-Y-[X]1-30-[R / K / Q / H]-[X]1-4-[S / T]-X-pwherein X is any residue, Y is tyrosine, S / T is serine or threonine and Ψ is a hydrophobic residue or an equivalent thereof. Preferably the residues are Tyr577 and Ser585 of the common βc of the GM-CSF / IL-5 / IL-3 receptor.

Compositions and methods are provided for the cell-mediated targeted killing of diseased cells based on the presence of an intracellular antigen, rather than a surface-bound marker. The targeting cells are modified to express a cytotoxic protein that is delivered into a targeted cell, and after delivery is selectively activated by the presence of a cytoplasmic protein of interest. In one embodiment of the invention, the cytotoxic molecule is a Granzyme B (GrB) polypeptide. In the compositions of the invention, GrB is modified to render its cytotoxic enzymatic functions inactive, until the presence of an intracellular antigen unlocks the GrB molecule to enable enzymatic activities.

The invention provides a reagent, preparation method and application for separating and extracting plasma protein and nucleoprotein in cells. The reagent includes lysate A: 6mM-12mM HEPES with a pH of 7.8, 9mM-11mM NaCl, 0.5 mM EDTA, 1mM- 2mM MgCl 2 ; Lysis Buffer B: 6mM‑12mM HEPES pH 7.8, pH 7.8, 9mM‑11mM NaCl, 0.5 mM EDTA, 1mM‑2mM MgCl 2 , 8%‑12% NP‑40; Lysis Solution C: 6mM‑12mM HEPES, pH 7.8, 9mM‑11mM NaCl, 0.5 mM EDTA, 1mM‑2mM MgCl 2 , 8%-12% glycerin, 8%-12% SDS. The invention can realize efficient, rapid and complete separation of cytoplasmic protein and nucleoprotein, and avoid cross contamination of the two.

This application is directed to methods which allow for encapsulation of an array of biological materials under physiological conditions that are relevant given a biological context and the compositions made using those methods. These reconstituted biological materials encompass: a) purified proteins that can bind to the lipid membranes inside and outside of vesicles based on the electrostatic charge; b) purified cytosolic proteins that position themselves in the lumen of the vesicles, c) mammalian cell extracts with an array of cytosolic protein content, among other contents and d) small biological molecules such as DNA and RNA as well as fluorescent dyes / probes. These vesicles can be used to simulate cells in drug discovery methods as well as useful in administering drugs and other compositions to cells in vitro and in vivo.

Embodiments of the invention find application in the field of cancer therapy. Receptor protein kinases (RPTKs) transmit extracellular signals across the plasma membrane to cytosolic proteins, stimulating formation of complexes that regulate key cellular functions. Over half of the known tyrosine kinases are implicated in human cancers and are therefore highly promising drug targets.

The present invention provides a method for producing large quantities of transmembrane and cytosolic proteins in a mammalian muscle cell. The method involves transfecting skeletal muscle cells in vivo with nucleic acids encoding the proteins by electroporation. The invention results in production of the desired protein on the order of about 2-3 magnitudes more as compared to standard methods, allowing for various biological uses including purification and crystallization.

Transmembrane B ephrins and their Eph receptors signal hi-directionally. The presently claimed invention describes a cytoplasmic protein, designated PDZ-RGS3, which binds B ephrins through a PDZ domain, and has a regulator of heterotrimeric G protein signaling (RGS) domain. PDZ-RGS3 mediates signaling from the ephrin-B cytoplasmic tail. SDF-1, a chemokine with a G protein coupled receptor, or BDNF, act as chemoattractants for cerebellar granule cells, with SDF-1 action being selectively inhibited by soluble EphB receptor. The claimed invention reveals a pathway that links reverse signaling to cellular guidance, uncovers a novel mode of control for G proteins, and demonstrates a mechanism for selective regulation of responsiveness to neuronal guidance cues. Further, compositions and methods of use are provided for modulating cell migration as a function of chemokines and GPCR interaction, to aid in the treatment of disease states and medical conditions, including cancer and immune responses such as allergy and autoimmune responses. In one embodiment, a method of altering the sensitivity of a cell to a chemokine is provided using a PDZ-RGS3 protein.

Methods of identifying agents which alter the NAD-dependent deacetylation activity of a Sir2 protein or a fragment of a Sir2 protein are disclosed. The acetylated protein can be a nuclear protein, such as a histone protein, or a cytoplasmic protein. The Sir2 protein employed in the methods can include at least a core domain of a Sir2 protein, such as a human Sir2 protein.