202 results about "Cytosol" patented technology

A high flux in conversion of pyruvate to acetolactate was achieved in yeast through expression of acetolactate synthase in the cytosol in conjunction with reduction in pyruvate decarboxylase activity. Additional manipulations to improve flux to acetolactate are reduced pyruvate dehydrogenase activity and reduced glycerol-3-phosphate dehydrogenase activity. Production of compounds having acetolactate as an upstream intermediate benefit from the increased conversion of pruvate to acetolactate in the described strains.

The present invention provides recombinant microorganisms comprising isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol, wherein said recombinant microorganism is selected to produce isobutanol from a carbon source. Methods of using said recombinant microorganisms to produce isobutanol are also provided. In various aspects of the invention, the recombinant microorganisms may comprise a cytosolically active isobutanol pathway enzymes. In some embodiments, the invention provides mutated, modified, and/or chimeric isobutanol pathway enzymes with cytosolic activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

A single chain, polypeptide fusion protein, comprising: a non-cytotoxic protease, or a fragment thereof, which protease or protease fragment is capable of cleaving a protein of the exocytic fusion apparatus of a nociceptive sensory afferent; a dynorphin Targeting Moiety that is capable of binding to a Binding Site on the nociceptive sensory afferent, which Binding Site is capable of undergoing endocytosis to be incorporated into an endosome within the nociceptive sensory afferent; a protease cleavage site at which site the fusion protein is cleavable by a protease, wherein the protease cleavage site is located between the non-cytotoxic protease or fragment thereof and the dynorphin Targeting Moiety; and a translocation domain that is capable of translocating the protease or protease fragment from within an endosome, across the endosomal membrane and into the cytosol of the nociceptive sensory afferent. Nucleic acid sequences encoding the polypeptide fusion proteins, methods of preparing same and uses thereof are also described.

Methods and compositions are provided for delivering a polynucleotide encoding a gene of interest to a target cell using a virus. The virus envelope comprises a cell-specific binding determinant that recognizes and binds to a component on the target cell surface, leading to endocytosis of the virus. A separate fusogenic molecule is also present on the envelope and facilitates delivery of the polynucleotide across the membrane and into the cytosol of the target cell. The methods and related compositions can be used for treating patients having suffering from a wide range of conditions, including infection, such as HIV; cancers, such as non-Hodgkin's lymphoma and breast cancer; and hematological disorders, such as severe combined immunodeficiency.

The present invention relates to a method of identifying a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in (the cytosol of) a yeast cell comprising: a) providing a mutated yeast cell comprising a deletion of at least one gene of the (PDH) by-pass, selected from the genes encoding the enzymes pyruvate decarboxylase (PDC), acetaldehyde dehydrogenase (ALD), and acetyl-CoA synthetase (ACS); b) transforming said mutated yeast cell with an expression vector comprising a heterologous nucleotide sequence encoding a candidate polypeptide having potential enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA; c) testing said recombinant mutated yeast cell for its ability to grow on minimal medium containing glucose as sole carbon source, and d) identifying said candidate polypeptide as a heterologous polypeptide having enzymatic activity for converting pyruvate, acetaldehyde or acetate into acetyl-CoA in (the cytosol of) said yeast cell when growth of said cell is observed. The invention further relates to a method of producing a fermentation production such as butanol.

A method for inhibiting therapeutic drug resistance within a cell over-expressing a membrane protein is provided. The method comprises synthesizing a dimeric prodrug inhibitor of a monomeric therapeutic agent; administering the dimeric prodrug inhibitor to the membrane protein together with the monomeric therapeutic agent; and occupying at least one substrate binding site of the membrane protein with the synthesized dimeric prodrug to allow the monomeric therapeutic agent to accumulate within the cell. The dimeric prodrug inhibitor contains a crosslinking agent that is adapted to breakdown under reducing conditions within the cytosol of the cell to cause the dimeric prodrug to revert back to a form equivalent to the monomeric therapeutic agent.

The present invention relates to a delivery system that comprises a conjugate that facilitates the delivery of a compound such as a biologically-active macromolecule, a nucleic acid or a peptide in particular, into a cell. The present invention also relates to said conjugate for delivery of a compound, such as a biologically-active macromolecule, a nucleic acid or a peptide, into a cell. The present invention further relates to a pharmaceutical composition comprising said conjugate and to its use. The present invention also relates to a method of delivering a compound to a cell or an organism, preferably a patient. The conjugates comprise: (a) at least one module that mediates cell targeting and facilitates cellular uptake, (b) at least one module that facilitates transport to the endoplasmic reticulum (ER), (c) at least one module that mediates translocation from the ER to the cytosol, and (d) at least one compound to be delivered wherein the modules (a) to (c) and the compound (d) are linked to each other in any arrangement.

The present invention relates to methods of making and using chimeric antibody molecules comprised of at least two domains, namely R_AF, and I_AF, linked by a flexible peptide linker.

Mycobacterial proteins from culture filtrate or cytosol are disclosed as being useful B cell antigens for early diagnosis of mycobacterial disease, particularly in humans. These proteins include four that had not previously been recognized as B cell antigens (LppZ protein encoded by Mtb gene Rv3006; SodC protein encoded by Mtb gene Rv0432; BfrB protein encoded by Mtb gene Rv3841 and TrxC protein encoded byMtb gene Rv3914). Antigenic compositions include these proteins and/or peptide fragments thereof, in various combinations with each other or with one or more of a set of 10 additional Mtb proteins known to be antigens (in paricular early antigens. Methods and kits for using these antigenic composition for early diagnosis of mycobacterial infection and disease are also disclosed.

A method and device for preferentially delivering a compound such as an antigen to the cytosol of an immune cell. The method comprises passing a cell suspension comprising the target immune cell through a microfluidic device and contacting the suspension with the compound(s) or payload to be delivered.

We disclose a recombinant yeast, wherein the yeast is pyruvate decarboxylase enzyme (PDC) activity negative (PDC-negative) and is functionally transformed with a coding region encoding a pyruvate carboxylase enzyme (PYC) wherein the PYC is active in the cytosol, a coding region encoding a malate dehydrogenase enzyme (MDH) wherein the MDH is active in the cytosol and is not inactivated in the presence of glucose, and a coding region encoding a malic acid transporter protein (MAE). We also disclose a method of producing malic acid by culturing such a yeast in a medium comprising a carbon source and a carbon dioxide source and isolating malic acid from the medium.

A compound comprising, in combination: a cell surface binding ligand or internalizing factor, such as an IL-13Rα2 binding ligand; at least one effector molecule (e.g., one, two, three or more effector molecules); optionally but preferably, a cytosol localization element covalently coupled between said binding ligand and said at least one effector molecule; and a subcellular compartment localization signal element covalently coupled between said binding ligand and said at least one effector molecule (and preferably with said cytosol localization element between said binding ligand and said subcellular compartment localization signal element). Methods of using such compounds and formulations containing the same are also described.

Disclosed herein are multimeric molecular complexes and compounds that are multivalent, i.e., they have two or more targeting elements directed to a ligand that confers paracellular transporting properties and/or transcytotic properties to complexes and compounds to which it is bound. The complexes and compounds have properties that are different from the properties of monomers, complexes and compounds having only one targeting element directed to a paracellular and/or transcytotic ligand. The complexes and compounds of the invention undergo endocytosis, transcytosis and exocytosis; following endocytosis, the complexes or compounds may be transported into the cytosol or an organelle of a cell. In polarized cells, transcytosis can proceed in a “forward” or “reverse” direction. Reverse transcytosis is used for the non-invasive delivery of biologically active agents from the lumen of, e.g., the gastrointestinal tract or the airways of lungs, to the circulatory system. The complexes and compounds are incorporated in various compositions and medical devices suitable for medicinal or veterinary use.

The invention relates to a microorganism as a carrier of nucleotide sequences coding for antigens and protein toxins comprising the following components: (I) at least one nucleotide sequence coding for at least one complete or partial antigen of at least one wild-type or mutated protein; and (II) at least one nucleotide sequence coding for at least one protein toxin and/or at least one protein toxin subunit; and (III) a) at least one nucleotide sequence coding for at least one transport system which enables the expression of the expression products of component (I) and component (II) on the outer surface of the microorganism and/or enables the secretion of the expression products of component (I) and component (II); and/or coding for at least one signal sequence which enables the secretion of the expression products of component (I) and component (II); and/or (III) b) optionally, at least one nucleotide sequence coding for at least one protein for lysing the microorganism in the cytosol of mammalian cells and for intracellularly releasing plasmids or expression vectors, which are contained in the lysed microorganism; and (IV) at least one nucleotide sequence for at least one activation sequence for the expression of one or more of components (I) to (III), wherein said activation sequence can be activated in the microorganism and/or is tissue cell-specific, tumor cell-specific, macrophage-specific, dendrite-specific, lymphocyte-specific, function-specific or non-cell-specific”; wherein any of components (I) to (IV) can be present either once or several times and either identical or different. Also disclosed are a process of manufacturing thereof, corresponding plasmids or expression vectors and uses of the microorganism as a medicament.

Methods for enhancing cellular uptake of cargo molecules by boronating the cargo molecule, particularly with one or more phenylboronic acid groups. Cellular uptake includes at least partial uptake into the cytosol. Boronation includes ligating, crosslinking or otherwise bonding one or more phenylboronic acids substituted to contain a reactive group to a cargo molecule. Boronation also includes ligating, crosslinking or otherwise bonding a phenylboronated oligopeptide to a cargo molecule. The phenylboronate groups are optionally conjugated to the cargo molecule via linking moieties that can be selectively cleaved, such cleavable linkers can allow the phenylboronate groups to be removed from the cargo molecule after the boronated cargo molecule is introduced into the cell. The invention includes certain phenylboronates which are boronation reagents, certain boronated oligopeptides and certain boronated peptides and proteins. The invention also includes kits for enhancing cellular uptake of cargo molecules by boronation with one or more phenylboronates or boronated oligopeptides.

The invention provides ion-selective sensors capable of selectively measuring ions, e.g., Na⁺, K⁺, Cl⁻, etc., in the cytosol of a single living cell. The sensor comprises one or more quantum dots or a fluorescent dye, a pH-sensitive dye, and optionally an ion-selective component such as an ionophore. These elements may, for example, be disposed in a polymer matrix. The polymer matrix comprises an internalizing moiety which enables the sensor to localize within the cytosol of a cell. The internalizing moiety comprises a small molecule or peptide such as an amine, antepennepedia, mastoparan, or melittin that react under acidic conditions to release a sensor from the confines of a endosome. Once in the cytosol the sensors may detect ionic analytes by selective ion extraction by the polymer, thereby inducing a pH change within the sensor which in turn changes the absorbance of the pH-sensitive dye. The change of absorbance may in turn attenuate the intensity of detectable emissions, e.g., fluorescence, from the quantum dot or dye by directly absorbing its fluorescence emission.

The invention provides ion-selective sensors comprising quantum dots capable of selectively measuring ions, e.g., Na⁺, K⁺, Cl⁻, etc., in various environments, including in the cytosol of a living cell. Quantum dots are attractive probes for microscopy due to their photophysical advantages over fluorescent dyes, including prolonged photostability, brightness and quantum efficiency. In certain embodiments, a sensor comprises one or more quantum dots, a pH-sensitive dye, and optionally an ion-selective component such as an ionophore. These elements may, for example, be disposed in a polymer matrix. In certain embodiments, the sensors may detect ionic analytes by selective ion extraction by the polymer, thereby inducing a pH change within the sensor which in turn changes the absorbance of the pH-sensitive dye. The change of absorbance may in turn attenuate the intensity of detectable emissions, e.g., fluorescence, from the quantum dot by directly absorbing its fluorescence emission.