Recombinant Fusion Proteins: Recombinant proteins produced by the GENETIC TRANSLATION of fused genes formed by the combination of NUCLEIC ACID REGULATORY SEQUENCES of one or more genes with the protein coding sequences of one or more genes.Molecular Sequence Data: Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.Amino Acid Sequence: The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.Recombinant Proteins: Proteins prepared by recombinant DNA technology.Cloning, Molecular: The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.Escherichia coli: A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.Base Sequence: The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence.Vaccines, Synthetic: Small synthetic peptides that mimic surface antigens of pathogens and are immunogenic, or vaccines manufactured with the aid of recombinant DNA techniques. The latter vaccines may also be whole viruses whose nucleic acids have been modified.Immunotoxins: Semisynthetic conjugates of various toxic molecules, including RADIOACTIVE ISOTOPES and bacterial or plant toxins, with specific immune substances such as IMMUNOGLOBULINS; MONOCLONAL ANTIBODIES; and ANTIGENS. The antitumor or antiviral immune substance carries the toxin to the tumor or infected cell where the toxin exerts its poisonous effect.Membrane Fusion: The adherence and merging of cell membranes, intracellular membranes, or artificial membranes to each other or to viruses, parasites, or interstitial particles through a variety of chemical and physical processes.Epitopes: Sites on an antigen that interact with specific antibodies.Viral Fusion Proteins: Proteins, usually glycoproteins, found in the viral envelopes of a variety of viruses. They promote cell membrane fusion and thereby may function in the uptake of the virus by cells.Cell Fusion: Fusion of somatic cells in vitro or in vivo, which results in somatic cell hybridization.Genetic Vectors: DNA molecules capable of autonomous replication within a host cell and into which other DNA sequences can be inserted and thus amplified. Many are derived from PLASMIDS; BACTERIOPHAGES; or VIRUSES. They are used for transporting foreign genes into recipient cells. Genetic vectors possess a functional replicator site and contain GENETIC MARKERS to facilitate their selective recognition.DNA, Complementary: Single-stranded complementary DNA synthesized from an RNA template by the action of RNA-dependent DNA polymerase. cDNA (i.e., complementary DNA, not circular DNA, not C-DNA) is used in a variety of molecular cloning experiments as well as serving as a specific hybridization probe.Immunoconjugates: Combinations of diagnostic or therapeutic substances linked with specific immune substances such as IMMUNOGLOBULINS; MONOCLONAL ANTIBODIES; or ANTIGENS. Often the diagnostic or therapeutic substance is a radionuclide. These conjugates are useful tools for specific targeting of DRUGS and RADIOISOTOPES in the CHEMOTHERAPY and RADIOIMMUNOTHERAPY of certain cancers.Cell Line: Established cell cultures that have the potential to propagate indefinitely.Protein Binding: The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.Sequence Homology, Amino Acid: The degree of similarity between sequences of amino acids. This information is useful for the analyzing genetic relatedness of proteins and species.Glutathione Transferase: A transferase that catalyzes the addition of aliphatic, aromatic, or heterocyclic FREE RADICALS as well as EPOXIDES and arene oxides to GLUTATHIONE. Addition takes place at the SULFUR. It also catalyzes the reduction of polyol nitrate by glutathione to polyol and nitrite.Antibodies, Monoclonal: Antibodies produced by a single clone of cells.Enzyme-Linked Immunosorbent Assay: An immunoassay utilizing an antibody labeled with an enzyme marker such as horseradish peroxidase. While either the enzyme or the antibody is bound to an immunosorbent substrate, they both retain their biologic activity; the change in enzyme activity as a result of the enzyme-antibody-antigen reaction is proportional to the concentration of the antigen and can be measured spectrophotometrically or with the naked eye. Many variations of the method have been developed.Gene Expression: The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION.Plasmids: Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.DNA Primers: Short sequences (generally about 10 base pairs) of DNA that are complementary to sequences of messenger RNA and allow reverse transcriptases to start copying the adjacent sequences of mRNA. Primers are used extensively in genetic and molecular biology techniques.Mice, Inbred BALB CBlotting, Western: Identification of proteins or peptides that have been electrophoretically separated by blot transferring from the electrophoresis gel to strips of nitrocellulose paper, followed by labeling with antibody probes.Binding Sites: The parts of a macromolecule that directly participate in its specific combination with another molecule.Immunoblotting: Immunologic method used for detecting or quantifying immunoreactive substances. The substance is identified by first immobilizing it by blotting onto a membrane and then tagging it with labeled antibodies.Electrophoresis, Polyacrylamide Gel: Electrophoresis in which a polyacrylamide gel is used as the diffusion medium.Bacterial Proteins: Proteins found in any species of bacterium.Protozoan Proteins: Proteins found in any species of protozoan.Antibody Specificity: The property of antibodies which enables them to react with some ANTIGENIC DETERMINANTS and not with others. Specificity is dependent on chemical composition, physical forces, and molecular structure at the binding site.Immunoglobulin G: The major immunoglobulin isotype class in normal human serum. There are several isotype subclasses of IgG, for example, IgG1, IgG2A, and IgG2B.Hepatocyte Nuclear Factor 3-beta: A forkhead transcription factor that regulates expression of metabolic GENES and is involved in EMBRYONIC DEVELOPMENT. Mutations in HNF-3beta have been associated with CONGENITAL HYPERINSULINISM.Diphtheria Toxin: An ADP-ribosylating polypeptide produced by CORYNEBACTERIUM DIPHTHERIAE that causes the signs and symptoms of DIPHTHERIA. It can be broken into two unequal domains: the smaller, catalytic A domain is the lethal moiety and contains MONO(ADP-RIBOSE) TRANSFERASES which transfers ADP RIBOSE to PEPTIDE ELONGATION FACTOR 2 thereby inhibiting protein synthesis; and the larger B domain that is needed for entry into cells.Membrane Glycoproteins: Glycoproteins found on the membrane or surface of cells.Antigens, Bacterial: Substances elaborated by bacteria that have antigenic activity.Tumor Cells, Cultured: Cells grown in vitro from neoplastic tissue. If they can be established as a TUMOR CELL LINE, they can be propagated in cell culture indefinitely.Polymerase Chain Reaction: In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships.Antibodies, Bacterial: Immunoglobulins produced in a response to BACTERIAL ANTIGENS.Autoantibodies: Antibodies that react with self-antigens (AUTOANTIGENS) of the organism that produced them.Oncogene Proteins, Fusion: The GENETIC TRANSLATION products of the fusion between an ONCOGENE and another gene. The latter may be of viral or cellular origin.Kinetics: The rate dynamics in chemical or physical systems.Molecular Weight: The sum of the weight of all the atoms in a molecule.Rabbits: The species Oryctolagus cuniculus, in the family Leporidae, order LAGOMORPHA. Rabbits are born in burrows, furless, and with eyes and ears closed. In contrast with HARES, rabbits have 22 chromosome pairs.Cattle: Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor.Gene Fusion: The GENETIC RECOMBINATION of the parts of two or more GENES resulting in a gene with different or additional regulatory regions, or a new chimeric gene product. ONCOGENE FUSION includes an ONCOGENE as at least one of the fusion partners and such gene fusions are often detected in neoplastic cells and are transcribed into ONCOGENE FUSION PROTEINS. ARTIFICIAL GENE FUSION is carried out in vitro by RECOMBINANT DNA technology.Spinal Fusion: Operative immobilization or ankylosis of two or more vertebrae by fusion of the vertebral bodies with a short bone graft or often with diskectomy or laminectomy. (From Blauvelt & Nelson, A Manual of Orthopaedic Terminology, 5th ed, p236; Dorland, 28th ed)T-Lymphocytes: Lymphocytes responsible for cell-mediated immunity. Two types have been identified - cytotoxic (T-LYMPHOCYTES, CYTOTOXIC) and helper T-lymphocytes (T-LYMPHOCYTES, HELPER-INDUCER). They are formed when lymphocytes circulate through the THYMUS GLAND and differentiate to thymocytes. When exposed to an antigen, they divide rapidly and produce large numbers of new T cells sensitized to that antigen.ADP Ribose Transferases: Enzymes that transfer the ADP-RIBOSE group of NAD or NADP to proteins or other small molecules. Transfer of ADP-ribose to water (i.e., hydrolysis) is catalyzed by the NADASES. The mono(ADP-ribose)transferases transfer a single ADP-ribose. POLY(ADP-RIBOSE) POLYMERASES transfer multiple units of ADP-ribose to protein targets, building POLY ADENOSINE DIPHOSPHATE RIBOSE in linear or branched chains.Protein Structure, Tertiary: The level of protein structure in which combinations of secondary protein structures (alpha helices, beta sheets, loop regions, and motifs) pack together to form folded shapes called domains. Disulfide bridges between cysteines in two different parts of the polypeptide chain along with other interactions between the chains play a role in the formation and stabilization of tertiary structure. Small proteins usually consist of only one domain but larger proteins may contain a number of domains connected by segments of polypeptide chain which lack regular secondary structure.Transfection: The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES.DNA, Recombinant: Biologically active DNA which has been formed by the in vitro joining of segments of DNA from different sources. It includes the recombination joint or edge of a heteroduplex region where two recombining DNA molecules are connected.Green Fluorescent Proteins: Protein analogs and derivatives of the Aequorea victoria green fluorescent protein that emit light (FLUORESCENCE) when excited with ULTRAVIOLET RAYS. They are used in REPORTER GENES in doing GENETIC TECHNIQUES. Numerous mutants have been made to emit other colors or be sensitive to pH.Peptide Fragments: Partial proteins formed by partial hydrolysis of complete proteins or generated through PROTEIN ENGINEERING techniques.Membrane Fusion Proteins: Proteins that catalyze MEMBRANE FUSION.Cells, Cultured: Cells propagated in vitro in special media conducive to their growth. Cultured cells are used to study developmental, morphologic, metabolic, physiologic, and genetic processes, among others.Luminescent Proteins: Proteins which are involved in the phenomenon of light emission in living systems. Included are the "enzymatic" and "non-enzymatic" types of system with or without the presence of oxygen or co-factors.Bacterial Toxins: Toxic substances formed in or elaborated by bacteria; they are usually proteins with high molecular weight and antigenicity; some are used as antibiotics and some to skin test for the presence of or susceptibility to certain diseases.Mutation: Any detectable and heritable change in the genetic material that causes a change in the GENOTYPE and which is transmitted to daughter cells and to succeeding generations.Cricetinae: A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS.Maltose-Binding Proteins: Periplasmic proteins that bind MALTOSE and maltodextrin. They take part in the maltose transport system of BACTERIA.Membrane Proteins: Proteins which are found in membranes including cellular and intracellular membranes. They consist of two types, peripheral and integral proteins. They include most membrane-associated enzymes, antigenic proteins, transport proteins, and drug, hormone, and lectin receptors.Baculoviridae: Family of INSECT VIRUSES containing two subfamilies: Eubaculovirinae (occluded baculoviruses) and Nudibaculovirinae (nonoccluded baculoviruses). The Eubaculovirinae, which contain polyhedron-shaped inclusion bodies, have two genera: NUCLEOPOLYHEDROVIRUS and GRANULOVIRUS. Baculovirus vectors are used for expression of foreign genes in insects.Viral Envelope Proteins: Layers of protein which surround the capsid in animal viruses with tubular nucleocapsids. The envelope consists of an inner layer of lipids and virus specified proteins also called membrane or matrix proteins. The outer layer consists of one or more types of morphological subunits called peplomers which project from the viral envelope; this layer always consists of glycoproteins.beta-Galactosidase: A group of enzymes that catalyzes the hydrolysis of terminal, non-reducing beta-D-galactose residues in beta-galactosides. Deficiency of beta-Galactosidase A1 may cause GANGLIOSIDOSIS, GM1.Recombination, Genetic: Production of new arrangements of DNA by various mechanisms such as assortment and segregation, CROSSING OVER; GENE CONVERSION; GENETIC TRANSFORMATION; GENETIC CONJUGATION; GENETIC TRANSDUCTION; or mixed infection of viruses.Carrier Proteins: Transport proteins that carry specific substances in the blood or across cell membranes.Cercopithecus aethiops: A species of CERCOPITHECUS containing three subspecies: C. tantalus, C. pygerythrus, and C. sabeus. They are found in the forests and savannah of Africa. The African green monkey (C. pygerythrus) is the natural host of SIMIAN IMMUNODEFICIENCY VIRUS and is used in AIDS research.Viral Proteins: Proteins found in any species of virus.Oncogene Fusion: The GENETIC RECOMBINATION of the parts of two or more GENES, including an ONCOGENE as at least one of the fusion partners. Such gene fusions are often detected in neoplastic cells and are transcribed into ONCOGENE FUSION PROTEINS.Mutagenesis, Site-Directed: Genetically engineered MUTAGENESIS at a specific site in the DNA molecule that introduces a base substitution, or an insertion or deletion.Protein Engineering: Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes.CHO Cells: CELL LINE derived from the ovary of the Chinese hamster, Cricetulus griseus (CRICETULUS). The species is a favorite for cytogenetic studies because of its small chromosome number. The cell line has provided model systems for the study of genetic alterations in cultured mammalian cells.Cell Membrane: The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.HeLa Cells: The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for VIRUS CULTIVATION and antitumor drug screening assays.Spodoptera: A genus of owlet moths of the family Noctuidae. These insects are used in molecular biology studies during all stages of their life cycle.DNA-Binding Proteins: Proteins which bind to DNA. The family includes proteins which bind to both double- and single-stranded DNA and also includes specific DNA binding proteins in serum which can be used as markers for malignant diseases.Transcription Factors: Endogenous substances, usually proteins, which are effective in the initiation, stimulation, or termination of the genetic transcription process.COS Cells: CELL LINES derived from the CV-1 cell line by transformation with a replication origin defective mutant of SV40 VIRUS, which codes for wild type large T antigen (ANTIGENS, POLYOMAVIRUS TRANSFORMING). They are used for transfection and cloning. (The CV-1 cell line was derived from the kidney of an adult male African green monkey (CERCOPITHECUS AETHIOPS).)Peptides: Members of the class of compounds composed of AMINO ACIDS joined together by peptide bonds between adjacent amino acids into linear, branched or cyclical structures. OLIGOPEPTIDES are composed of approximately 2-12 amino acids. Polypeptides are composed of approximately 13 or more amino acids. PROTEINS are linear polypeptides that are normally synthesized on RIBOSOMES.Virus Internalization: The entering of cells by viruses following VIRUS ATTACHMENT. This is achieved by ENDOCYTOSIS, by direct MEMBRANE FUSION of the viral membrane with the CELL MEMBRANE, or by translocation of the whole virus across the cell membrane.Transcription, Genetic: The biosynthesis of RNA carried out on a template of DNA. The biosynthesis of DNA from an RNA template is called REVERSE TRANSCRIPTION.Protein Conformation: The characteristic 3-dimensional shape of a protein, including the secondary, supersecondary (motifs), tertiary (domains) and quaternary structure of the peptide chain. PROTEIN STRUCTURE, QUATERNARY describes the conformation assumed by multimeric proteins (aggregates of more than one polypeptide chain).RNA, Messenger: RNA sequences that serve as templates for protein synthesis. Bacterial mRNAs are generally primary transcripts in that they do not require post-transcriptional processing. Eukaryotic mRNA is synthesized in the nucleus and must be exported to the cytoplasm for translation. Most eukaryotic mRNAs have a sequence of polyadenylic acid at the 3' end, referred to as the poly(A) tail. The function of this tail is not known for certain, but it may play a role in the export of mature mRNA from the nucleus as well as in helping stabilize some mRNA molecules by retarding their degradation in the cytoplasm.Immunoglobulin Fc Fragments: Crystallizable fragments composed of the carboxy-terminal halves of both IMMUNOGLOBULIN HEAVY CHAINS linked to each other by disulfide bonds. Fc fragments contain the carboxy-terminal parts of the heavy chain constant regions that are responsible for the effector functions of an immunoglobulin (COMPLEMENT fixation, binding to the cell membrane via FC RECEPTORS, and placental transport). This fragment can be obtained by digestion of immunoglobulins with the proteolytic enzyme PAPAIN.Restriction Mapping: Use of restriction endonucleases to analyze and generate a physical map of genomes, genes, or other segments of DNA.Substrate Specificity: A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.Saccharomyces cerevisiae: A species of the genus SACCHAROMYCES, family Saccharomycetaceae, order Saccharomycetales, known as "baker's" or "brewer's" yeast. The dried form is used as a dietary supplement.Translocation, Genetic: A type of chromosome aberration characterized by CHROMOSOME BREAKAGE and transfer of the broken-off portion to another location, often to a different chromosome.Pichia: Yeast-like ascomycetous fungi of the family Saccharomycetaceae, order SACCHAROMYCETALES isolated from exuded tree sap.Microscopy, Fluorescence: Microscopy of specimens stained with fluorescent dye (usually fluorescein isothiocyanate) or of naturally fluorescent materials, which emit light when exposed to ultraviolet or blue light. Immunofluorescence microscopy utilizes antibodies that are labeled with fluorescent dye.Genes, Bacterial: The functional hereditary units of BACTERIA.DNA: A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine).Mice, Inbred C57BLSequence Deletion: Deletion of sequences of nucleic acids from the genetic material of an individual.Vesicular Transport Proteins: A broad category of proteins involved in the formation, transport and dissolution of TRANSPORT VESICLES. They play a role in the intracellular transport of molecules contained within membrane vesicles. Vesicular transport proteins are distinguished from MEMBRANE TRANSPORT PROTEINS, which move molecules across membranes, by the mode in which the molecules are transported.Aquaporin 1: Aquaporin 1 forms a water-specific channel that is constitutively expressed at the PLASMA MEMBRANE of ERYTHROCYTES and KIDNEY TUBULES, PROXIMAL. It provides these cells with a high permeability to WATER. In humans polymorphisms of this protein result in the Colton blood group antigen.Models, Molecular: Models used experimentally or theoretically to study molecular shape, electronic properties, or interactions; includes analogous molecules, computer-generated graphics, and mechanical structures.Antibodies, Viral: Immunoglobulins produced in response to VIRAL ANTIGENS.Protein Transport: The process of moving proteins from one cellular compartment (including extracellular) to another by various sorting and transport mechanisms such as gated transport, protein translocation, and vesicular transport.Giant Cells: Multinucleated masses produced by the fusion of many cells; often associated with viral infections. In AIDS, they are induced when the envelope glycoprotein of the HIV virus binds to the CD4 antigen of uninfected neighboring T4 cells. The resulting syncytium leads to cell death and thus may account for the cytopathic effect of the virus.Vero Cells: A CELL LINE derived from the kidney of the African green (vervet) monkey, (CERCOPITHECUS AETHIOPS) used primarily in virus replication studies and plaque assays.

VEGF is required for growth and survival in neonatal mice. (1/41883)

We employed two independent approaches to inactivate the angiogenic protein VEGF in newborn mice: inducible, Cre-loxP- mediated gene targeting, or administration of mFlt(1-3)-IgG, a soluble VEGF receptor chimeric protein. Partial inhibition of VEGF achieved by inducible gene targeting resulted in increased mortality, stunted body growth and impaired organ development, most notably of the liver. Administration of mFlt(1-3)-IgG, which achieves a higher degree of VEGF inhibition, resulted in nearly complete growth arrest and lethality. Ultrastructural analysis documented alterations in endothelial and other cell types. Histological and biochemical changes consistent with liver and renal failure were observed. Endothelial cells isolated from the liver of mFlt(1-3)-IgG-treated neonates demonstrated an increased apoptotic index, indicating that VEGF is required not only for proliferation but also for survival of endothelial cells. However, such treatment resulted in less significant alterations as the animal matured, and the dependence on VEGF was eventually lost some time after the fourth postnatal week. Administration of mFlt(1-3)-IgG to juvenile mice failed to induce apoptosis in liver endothelial cells. Thus, VEGF is essential for growth and survival in early postnatal life. However, in the fully developed animal, VEGF is likely to be involved primarily in active angiogenesis processes such as corpus luteum development.  (+info)

Cell polarization: chemotaxis gets CRACKing. (2/41883)

An early stage in the establishment of cell polarity during chemotaxis of Dictyostelium dicoideum has been identified by a recent study; the new results also show that the development of cell polarity does not rely upon cytoskeletal rearrangement, and may use a spatial sensing mechanism.  (+info)

Deletion analysis of the Drosophila Inscuteable protein reveals domains for cortical localization and asymmetric localization. (3/41883)

The Drosophila Inscuteable protein acts as a key regulator of asymmetric cell division during the development of the nervous system [1] [2]. In neuroblasts, Inscuteable localizes into an apical cortical crescent during late interphase and most of mitosis. During mitosis, Inscuteable is required for the correct apical-basal orientation of the mitotic spindle and for the asymmetric segregation of the proteins Numb [3] [4] [5], Prospero [5] [6] [7] and Miranda [8] [9] into the basal daughter cell. When Inscuteable is ectopically expressed in epidermal cells, which normally orient their mitotic spindle parallel to the embryo surface, these cells reorient their mitotic spindle and divide perpendicularly to the surface [1]. Like the Inscuteable protein, the inscuteable RNA is asymmetrically localized [10]. We show here that inscuteable RNA localization is not required for Inscuteable protein localization. We found that a central 364 amino acid domain - the Inscuteable asymmetry domain - was necessary and sufficient for Inscuteable localization and function. Within this domain, a separate 100 amino acid region was required for asymmetric localization along the cortex, whereas a 158 amino acid region directed localization to the cell cortex. The same 158 amino acid fragment could localize asymmetrically when coexpressed with the full-length protein, however, and could bind to Inscuteable in vitro, suggesting that this domain may be involved in the self-association of Inscuteable in vivo.  (+info)

Bcl-2 regulates amplification of caspase activation by cytochrome c. (4/41883)

Caspases, a family of specific proteases, have central roles in apoptosis [1]. Caspase activation in response to diverse apoptotic stimuli involves the relocalisation of cytochrome c from mitochondria to the cytoplasm where it stimulates the proteolytic processing of caspase precursors. Cytochrome c release is controlled by members of the Bcl-2 family of apoptosis regulators [2] [3]. The anti-apoptotic members Bcl-2 and Bcl-xL may also control caspase activation independently of cytochrome c relocalisation or may inhibit a positive feedback mechanism [4] [5] [6] [7]. Here, we investigate the role of Bcl-2 family proteins in the regulation of caspase activation using a model cell-free system. We found that Bcl-2 and Bcl-xL set a threshold in the amount of cytochrome c required to activate caspases, even in soluble extracts lacking mitochondria. Addition of dATP (which stimulates the procaspase-processing factor Apaf-1 [8] [9]) overcame inhibition of caspase activation by Bcl-2, but did not prevent the control of cytochrome c release from mitochondria by Bcl-2. Cytochrome c release was accelerated by active caspase-3 and this positive feedback was negatively regulated by Bcl-2. These results provide evidence for a mechanism to amplify caspase activation that is suppressed at several distinct steps by Bcl-2, even after cytochrome c is released from mitochondria.  (+info)

Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral activation. (5/41883)

In COS cells, Ral GDP dissociation stimulator (RalGDS)-induced Ral activation was stimulated by RasG12V or a Rap1/Ras chimera in which the N-terminal region of Rap1 was ligated to the C-terminal region of Ras but not by Rap1G12V or a Ras/Rap1 chimera in which the N-terminal region of Ras was ligated to the C-terminal region of Rap1, although RalGDS interacted with these small GTP-binding proteins. When RasG12V, Ral and the Rap1/Ras chimera were individually expressed in NIH3T3 cells, they localized to the plasma membrane. Rap1Q63E and the Ras/Rap1 chimera were detected in the perinuclear region. When RalGDS was expressed alone, it was abundant in the cytoplasm. When coexpressed with RasG12V or the Rap1/Ras chimera, RalGDS was detected at the plasma membrane, whereas when coexpressed with Rap1Q63E or the Ras/Rap1 chimera, RalGDS was observed in the perinuclear region. RalGDS which was targeted to the plasma membrane by the addition of Ras farnesylation site (RalGDS-CAAX) activated Ral in the absence of RasG12V. Although RalGDS did not stimulate the dissociation of GDP from Ral in the absence of the GTP-bound form of Ras in a reconstitution assay using the liposomes, RalGDS-CAAX could stimulate it without Ras. RasG12V activated Raf-1 when they were coexpressed in Sf9 cells, whereas RasG12V did not affect the RalGDS activity. These results indicate that Ras recruits RalGDS to the plasma membrane and that the translocated RalGDS induces the activation of Ral, but that Rap1 does not activate Ral due to distinct subcellular localization.  (+info)

B-MYB transactivates its own promoter through SP1-binding sites. (6/41883)

B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family.  (+info)

A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA. (7/41883)

The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.  (+info)

Assembly requirements of PU.1-Pip (IRF-4) activator complexes: inhibiting function in vivo using fused dimers. (8/41883)

Gene expression in higher eukaryotes appears to be regulated by specific combinations of transcription factors binding to regulatory sequences. The Ets factor PU.1 and the IRF protein Pip (IRF-4) represent a pair of interacting transcription factors implicated in regulating B cell-specific gene expression. Pip is recruited to its binding site on DNA by phosphorylated PU.1. PU.1-Pip interaction is shown to be template directed and involves two distinct protein-protein interaction surfaces: (i) the ets and IRF DNA-binding domains; and (ii) the phosphorylated PEST region of PU.1 and a lysine-requiring putative alpha-helix in Pip. Thus, a coordinated set of protein-protein and protein-DNA contacts are essential for PU.1-Pip ternary complex assembly. To analyze the function of these factors in vivo, we engineered chimeric repressors containing the ets and IRF DNA-binding domains connected by a flexible POU domain linker. When stably expressed, the wild-type fused dimer strongly repressed the expression of a rearranged immunoglobulin lambda gene, thereby establishing the functional importance of PU.1-Pip complexes in B cell gene expression. Comparative analysis of the wild-type dimer with a series of mutant dimers distinguished a gene regulated by PU.1 and Pip from one regulated by PU.1 alone. This strategy should prove generally useful in analyzing the function of interacting transcription factors in vivo, and for identifying novel genes regulated by such complexes.  (+info)

  • After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. (biomedcentral.com)
  • To obtain a high expressing clone of P. pastoris to produce recombinant Hv1a/GNA, a straightforward method was used to produce multi-copy expression plasmids, which does not require multiple integrations to give clones of P. pastoris containing high copy numbers of the introduced gene. (springer.com)
  • To prevent proteolysis, the Hv1a/GNA fusion expression construct was modified by site-directed mutagenesis to remove a potential Kex2 cleavage site at the C-terminus of the Hv1a peptide. (springer.com)
  • Fitches EC, Pyati PS, King GF, Gatehouse JA (2012) Fusion to snowdrop lectin dramatically enhances the oral activity of the insecticidal peptide ω-hexatoxin-Hv1a by mediating its delivery to the central nervous system. (springer.com)
  • An alternative strategy was also adopted with a view to stapling a recombinant BoNT/AΔHCC-SNAP-25 protein (without the neuronal binding domain) with Vamp2.βNGF produced in E. coli, and a synthetic syntaxin-1 peptide via SNARE complex formation. (dcu.ie)
  • Encouragingly, after inclusion of this leader peptide sequence into a Vamp2.βNGF construct, it was expressed as an active protein, although with low yield. (dcu.ie)
  • ORCID: https://orcid.org/0000-0002-3882-6129 2016, Novel fusion proteins based on recombinant lectins for delivery of a cytotoxic peptide specifically to cancer cells , PhD thesis, University of Salford. (salford.ac.uk)
  • The short peptide was expressed directly as a fusion protein with a superfolder form of the green florescent protein ( sf GFP), resulting in a high yield expression of soluble sf GFP-mutacin fusion protein (30 kDa) in the cytoplasm of E. coli. (openmicrobiologyjournal.com)
  • This is the first report of three different fusion proteins with an antitumor-analgesic peptide obtained from Chinese scorpion Buthus martensii Karsch (BmKAGAP). (bvsalud.org)
  • Elastin‐like polypeptides (ELPs) are recombinant peptide‐based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. (ingentaconnect.com)
  • In the phase 3 B-LONG (Recombinant Factor IX Fc Fusion Protein [rFIXFc] in Subjects With Haemophilia B) study, rFIXFc demonstrated a prolonged half-life compared with recombinant factor IX (rFIX), and safety and efficacy for prophylaxis and treatment of bleeding in subjects with moderately-severe to severe haemophilia B. In this B-LONG sub-analysis, rFIXFc was evaluated for efficacy in subjects requiring major surgery. (elsevier.com)
  • The formation of this vaccine construction was accompanied by significant conformational changes in the chimeric protein. (mdpi.com)
  • We have previously developed a fusion protein with 3 copies of the ectodomain of matrix protein 2 (M2e), which is one of the most explored conserved influenza A virus antigens for a broadly protective vaccine known today. (frontiersin.org)
  • Findings in this and our earlier studies define the bivalent fusion protein rVE as a potent candidate vaccine molecule with the capability to concurrently stimulate humoral and cell mediated immune responses and a proof of concept for developing efficient subunit vaccines against Gram negative facultative intracellular bacterial pathogens. (uni-wuerzburg.de)
  • In this study, we evaluated a recombinant vaccine prepared from non-pathogenic Mycobacterium smegmatis (rMS) that expresses a fusion of early secreted antigenic target 6-kDa antigen (ESAT6) and culture filtrate protein 10 (CFP10). (semanticscholar.org)
  • METHODS: We compared the efficacies of the most commonly used vaccine constructs--adjuvanted protein, plasmid DNA, and live bacterial vectors--bearing the immunodominant secreted antigens early secreted antigen target-6 and antigen 85B, either alone or as a fusion protein. (mendeley.com)
  • This study was undertaken to determine whether the addition of etanercept, a soluble tumor necrosis factor receptor (p75):Fc fusion protein (TNFR:Fc), to methotrexate therapy would provide additional benefit to patients who had persistent rheumatoid arthritis despite receiving methotrexate. (nih.gov)
  • The aim of this study was to develop a strategy for pain management by generating a biotherapeutic that requires targeting of soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) within the hyper-active sensory nerves. (dcu.ie)
  • Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. (biomedcentral.com)
  • Alternative splicing of downstream exons of the GNAS gene is observed, which results in different forms of the stimulatory G protein alpha subunit, a key element of the classical signal transduction pathway linking receptor-ligand interactions with the activation of adenylyl cyclase and a variety of cellular reponses. (abcam.com)
  • The recombinant protein was produced by transient gene expression (TGE) in suspension-adapted human embryonic kidney cells (HEK-293E) in 4L orbitally shaken bioreactors. (epfl.ch)
  • To overcome this problem we attached a signal sequence, as well as an amino-terminal His-tag fusion to the GM-CSF gene. (springer.com)
  • The sequence data showed a clear indication that the insert is our desired gene product allowing us to go on to design a fusion protein. (salford.ac.uk)
  • Its expression is induced by hypoxia and its role may be to protect hypoxic cells from apoptosis.This gene encodes a protein-disulfide isomerase. (antibody-antibodies.com)
  • After N-terminal amino acid sequence analysis, PCR-based strategy was used to clone the gene that encodes this protein. (pnas.org)
  • Nonetheless, from the clinical standpoint, gene transfer has limitations, including toxicity, limited control of the levels of protein expression, and difficulty in expressing more than one protein at a time. (jimmunol.org)
  • Protein transfer (or protein "painting"), which enables simultaneous delivery of well-defined amounts of multiple proteins to a cell, provides a valuable alternative means to gene transfer ( 9 , 10 ). (jimmunol.org)
  • Identifying proteins selectively associated with a genomic locus provides an important entry point toward understanding how a specific gene is regulated. (pnas.org)
  • Eukaryotic gene regulation is a complex process, often coordinated by the action of tens to hundreds of proteins. (pnas.org)
  • To fully understand the molecular mechanisms governing multiple steps in gene expression, including transcription and posttranscriptional regulation for a given gene, one must first identify the various protein factors involved in the process. (pnas.org)
  • F gene open reading frame (ORF) from NDV is 1650 bp, encoding a protein of 553 amino acids that can induce protective immunity alone. (org.ir)
  • Shafaati M R, Moghbeli M, Dorostkar R. Construction of Recombinant Bacmid DNA Encoding Newcastle Disease Virus (NDV) Fusion Protein Gene. (org.ir)
  • Plasminogen-related protein B is a protein that in humans is encoded by the PLGLB2 gene. (wikipedia.org)
  • We have generated antibody fusion proteins directed against HER2, linked to sequences encoding an NKG2D-L, which can directly activate cytotoxic function in NK cells and also costimulate cytotoxic T cells. (aacrjournals.org)
  • A large amount of the recombinant fusion proteins with high purity was obtained by applying a single step anion exchange chromatography or metal chelate affinity. (jascoinc.com)
  • We analyzed the blood loss patterns of 617 consecutive adult patients undergoing lumbar and/or thoracic fusions requiring subfascial drain placement at a single institution from January 2009 to December 2016. (asianspinejournal.org)
  • To independently assess the effectiveness and harms of rhBMP-2 in spinal fusion and reporting bias in industry-sponsored journal publications. (nih.gov)
  • In spinal fusion, rhBMP-2 has no proven clinical advantage over bone graft and may be associated with important harms, making it difficult to identify clear indications for rhBMP-2. (nih.gov)
  • The authors conclude that on the basis of the currently available evidence, it is difficult to identify clear indications for rhBMP-2 in spinal fusion. (spinesection.org)
  • Medtronic Sofamor Danek, Memphis, TN, USA) has been used as an osteoinductive adjuvant to enhance spinal fusions for more than 15 years [ 1 - 3 ]. (asianspinejournal.org)
  • Therefore, in this study, we aimed to use a retrospective cohort design to examine the impact of rhBMP-2 on the total perioperative blood loss (intraoperative estimated blood loss and postoperative drain output) following lumbar and thoracic spinal fusions involving the placement of a subfascial drain. (asianspinejournal.org)
  • Our study sought to empirically test the hypothesis that the use of rhBMP-2 during spinal fusion procedures reduces intraoperative and postoperative blood loss relative to the use of ICBG. (asianspinejournal.org)
  • We hypothesized that a high dose of rhBMP-2 in cervical spinal fusion could induce substantial alterations in bone, leading to mechanical impairment. (scienceopen.com)
  • Biologic manipulation for spinal fusion is an area undergoing active research. (eduhk.hk)
  • For this study, 24 adult New Zealand white rabbits underwent single-level unilateral posterior intertransverse process spinal fusion at L5-L6. (eduhk.hk)
  • In this study we looked into the possible reasons for poor expression and found that protein toxicity coupled with protease-based degradation was the principal reason for low productivity. (springer.com)
  • This study presents an approach for inhibiting p53-deficient tumor cell growth by using protein-based E4orf4 that had been genetically fused to epidermal growth factor (EGF) to ensure selective targeting of EGF receptor-overexpressing tumor cells. (ovid.com)
  • Included in this study are the first recombinant PRV strains expressing C terminal VP26-mCherry. (princeton.edu)
  • In our previous study, we established that the bivalent fusion protein (rVE) comprising immunologically active regions of Y pestis LcrV (100-270 aa) and YopE (50-213 aa) proteins conferred complete passive and active protection against lethal Y enterocolitica 8081 challenge. (uni-wuerzburg.de)
  • Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B . New England Journal of Medicine , 369 (24), 2313-2323. (elsevier.com)
  • Treatment of bleeding episodes with recombinant factor VIII Fc fusion protein in A-LONG study subjects with severe haemophilia A. (qmul.ac.uk)
  • Consequently, in this study, we delivered intratumorally via protein transfer four molecules, including the chemotactic molecules secondary lymphoid tissue chemokine and Fas ligand and the costimulatory molecules 4-1BBL and TNF-related activation-induced cytokine. (jimmunol.org)
  • A prospective randomized study of posterolateral lumbar fusion using osteogenic protein-1 (OP-1) versus local autograft with ceramic bone substitute: emphasis of surgical exploration and histologic assessment. (scienceopen.com)
  • This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides. (ingentaconnect.com)
  • IDELVION is contraindicated in patients who have had life-threatening hypersensitivity to the product or its components, including hamster proteins. (idelvion.com)
  • For lumbar spine fusion, rhBMP-2 and iliac crest bone graft were similar in overall success, fusion, and other effectiveness measures and in risk for any adverse event, although rates were high across interventions (77% to 93% at 24 months from surgery). (nih.gov)
  • For both lumbar and cervical fusions, rhBMP-2 and iliac crest bone graft (ICBG) had similar results for fusion. (spinesection.org)
  • Previous studies on rhBMP-2 versus iliac crest bone grafting in thoracic and lumbar fusions have yielded mixed results regarding reductions in blood loss and have largely neglected the postoperative period when analyzing total blood loss. (asianspinejournal.org)
  • Since then, many studies have reported reduction in the operative time and hospital stays, as well as improved fusion rates in patients treated with rhBMP-2 compared with those treated with autologous iliac crest bone grafting (ICBG) [ 3 , 4 ]. (asianspinejournal.org)
  • n = 8/group) was used to quantify the consequences of a high rhBMP-2 dose (6 mg rhBMP-2) on fusion tissue compared to the 'gold standard' of autologous, cancellous bone graft. (scienceopen.com)
  • Although high-dose rhBMP-2 treatment led to an advanced radiological fusion result compared to autograft treatment, heterotopic bone formation and vertebral bone resorption were induced simultaneously. (scienceopen.com)
  • Histological evaluation unveiled highly active bone-forming processes ventral to the fusion segment after 12 weeks, while radiolucent areas showed still a partial loss of regular trabecular structure, with rare signs of remodelling and restoration. (scienceopen.com)
  • Despite qualitative alteration of the trabecular bone structure within the fusion site, the massive anterior heterotopic bone formation led to a substantial increase in mechanical stiffness compared to the autograft group. (scienceopen.com)
  • In 1965, Urist 14) discovered a subset of protein extract, which had a significant potential of inducing new bone formation even at the non-osseous tissues. (e-neurospine.org)
  • The proteins were termed bone morphogenetic proteins (BMPs). (e-neurospine.org)
  • 4) and in their letter they proposed that the containment of low dose (1.05 mg) rhBMP-2 within the cage and placing demineralized bone matrix (DBM) putty surrounding the INFUSE ensures a controlled delivery of rhBMP-2 in the vicinity of desired fusion site, which therefore is associated with excellent fusion rates with no adverse effects related to rhBMP-2. (e-neurospine.org)
  • Fitches EC, Bell HA, Powell ME, Back E, Sargiotti C, Weaver RJ, Gatehouse JA (2010) Insecticidal activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders. (springer.com)
  • These scFv were genetically fused with dimerization domains as well as with alkaline phosphatase, respectively, and diagnostic reagents were produced by expressing these fusion proteins in bacterial cultures. (gva.es)
  • Can Multi-Stage Recombinant Fusion Proteins be Considered as Reliable Vaccines Against Tuberculosis? (mcjbums.com)
  • Keikha M, Karbalaei M. Can Multi-Stage Recombinant Fusion Proteins be Considered as Reliable Vaccines Against Tuberculosis? (mcjbums.com)
  • Likewise, the relative immunodominance of individual antigens in the fusion molecule is altered by the choice of delivery system. (mendeley.com)
  • Recombinant factor VIII Fc fusion protein: extended-interval dosing maintains low bleeding rates and correlates with von Willebrand factor levels. (eloctate.com)
  • Just as the blades of a windmill harness naturally occurring wind to produce energy, Fc Fusion utilizes naturally occurring Fc receptors in your body to keep Factor VIII temporarily recirculating in your bloodstream, protecting you from bleeds. (eloctate.com)
  • Therefore, surface proteins of M. leprae cell wall that bind laminin-2 are crucial for bacterial targeting to the peripheral nerves. (pnas.org)
  • Here, we applied our method to isolate the Drosophila melanogaster histone cluster in S2 cells to identify several factors including Vig and Vig2, two proteins that bind and regulate core histone H2A and H3 mRNA via interaction with their 3′ UTRs. (pnas.org)